Devices and methods for cell analysis

ABSTRACT

The disclosure provides devices, device systems, and methods for analyzing cells (e.g., blood cells) or particles in a sample. In some embodiments, the disclosure provides various devices and device systems including: a light source; a collecting lens; and one, two, or more detectors. In other embodiments, the devices and device systems include a flow cell or a cartridge device with a flow cell. In further embodiments, the disclosure provides various methods including the steps of: using a light source to emit an irradiation light; using the irradiation light to illuminate a sample flow; using a collecting lens to collect both scattered light and fluorescent light from the sample flow; and using one, two, or more detectors to detect the collected scattered light and fluorescent light. Optionally, these methods include using a flow cell to form a sample flow.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the United States national stage entry under 35U.S.C. 371 of PCT/US2018/037114 filed on Jun. 12, 2018, and furtherclaims priority to U.S. Provisional Patent Application No. 62/519,467filed on Jun. 14, 2017, the disclosure of which are incorporated byreference herein in their entireties.

FIELD OF THE DISCLOSURE

The disclosure relates generally to the field of medicine, cytometry,and medical devices. More specifically, the disclosure relates to thefield of medical devices and methods for cell analysis.

BACKGROUND

All publications cited herein are incorporated by reference in theirentirety to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The following description includesinformation that may be useful in understanding the present disclosure.It is not an admission that any of the information provided herein isprior art or relevant to the presently disclosure, or that anypublication specifically or implicitly referenced is prior art.

Flow cytometry is a powerful method for detecting cells in a sample andanalyzing their characteristics with high throughput. By forming asample flow in a flow cell and irradiating the sample flow with lightfrom a light source, signals such as scattered light in forward angle,scatted light in side angle, and fluorescence can be detected fromindividual cells, and can be used to analyze their characteristics suchas cell size, cell granularity, cellular nucleic acids, cellularmembrane integrity, and cellular antigen expressions, etc.

In clinical applications, flow cytometry has been widely used fordetecting and analyzing cells in human or animal blood, such asenumerating the number of blood cells; classifying blood cells intodiverse types (e.g., white blood cells, red blood cells and plateletcells); and analyzing cellular antigen expressions (e.g., CD4⁺ antigen,CD8⁺ antigen, etc.). For example, in hematology analysis, flow cytometryhas been used to measure the total counts of white blood cells, redblood cells and platelet cells per sample volume, and to furtherclassify white blood cells into different subtypes (e.g., lymphocytes,monocytes, neutrophils, eosinophils, and basophils) and determine theirrespective percentages. For another example, flow cytometry is used inAIDS diagnostics for counting the number of CD4⁺ lymphocyte cells andCD8⁺ lymphocyte cells in blood.

Traditional analyzers for flow cytometry analysis normally have a fixedflow cell to form the sample flow. The alignment between the flow celland optical detection components is fixed and has no deviation. On thecontrary, in analyzers having a replaceable or disposable flow cell, thealignment between the flow cell and optical detection components mayhave significant deviation when the flow cell is replaced each time.This alignment deviation becomes a prominent issue for analyzers inwhich the flow cell is disposed and replaced after each samplemeasurement.

Furthermore, the flow cell in traditional analyzers normally has atleast two optical transparent surfaces for signal detection, where onesurface is used for measuring scattered light with a forward angle(e.g., a scattering angle less than about 20 degrees) from the sampleflow and the other surface is used for measuring scattered light with aside angle (e.g., a scattering angle more than about 70 degrees),fluorescence, or both from the sample flow. However, in some analyzers,the flow cell (e.g., some low-cost flow cells made by plastic injectionmolding process) may have only one optical transparent surface forsignal detection. Low-cost replaceable or disposable flow cells arenecessary for many applications such as point-of-care diagnostics. Butflow cells having only one optical transparent surface for signaldetection may limit the options of detectable signals.

Additionally, the replaceable or disposable flow cell is normally builtinto a cartridge device, and the cartridge device's surfaces or anyother surface in the optical path can reflect light back into the lightsource and introduce undesired noise. Also, the intensity of the lightsignals detected from a target (e.g., particles and cells) in thereplaceable or disposable flow cell can be weak, and hence it ischallenging to improve the collection efficiency of the light signals.

To address these challenges, the present disclosure provides variousdevices and methods for analyzing particles and cells.

SUMMARY

The following presents a simplified summary of the invention in order toprovide a basic understanding of some aspects of the invention. Thissummary is not an extensive overview of the invention. It is notintended to identify critical elements or to delineate the scope of theinvention. Its sole purpose is to present some concepts of the inventionin a simplified form as a prelude to the more detailed description thatis presented elsewhere.

To address those challenges as discussed above, the present disclosureprovides various devices and methods for analyzing particles and cells.In various embodiments, this disclosure provides various devices andmethods for analyzing cells in blood samples. In various embodiments,these device and methods can also be used for analyzing other particles(e.g., beads, nanoparticles, protein molecules, nucleic acid molecules,etc.) in a sample. In various embodiments, these devices and methods aresuitable for replaceable or disposable flow cells. In variousembodiments, these devices and methods are suitable for flow cellshaving only one optical transparent surface for signal detection andmeasurement. In various embodiments, these devices and methods are alsocompatible for other flow cells, for example, fixed flow cells and flowcells having more than one optical transparent surface for signaldetection and measurement.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a light source configured to emit an irradiationlight for illuminating a sample flow, wherein the sample flow comprisesparticles and/or cells; a collecting lens configured to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aforward angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a flow cell configured to form a sample flow of ameasurement sample, wherein the measurement sample comprises particlesand/or cells; a light source configured to emit an irradiation light forilluminating the sample flow; a collecting lens configured to collectboth a scattered light with a forward angle and a fluorescent light fromthe particles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aforward angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a sample flow, comprising: using alight source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a forward angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a measurement sample, comprising:using a flow cell to form a sample flow of the measurement sample; usinga light source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a forward angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a light source configured to emit an irradiationlight for illuminating a sample flow, wherein the sample flow comprisesparticles and/or cells; a collecting lens configured to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aside angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a flow cell configured to form a sample flow of ameasurement sample, wherein the measurement sample comprises particlesand/or cells; a light source configured to emit an irradiation light forilluminating the sample flow; a collecting lens configured to collectboth a scattered light with a side angle and a fluorescent light fromthe particles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aside angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a sample flow, comprising: using alight source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a side angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a measurement sample, comprising:using a flow cell to form a sample flow of the measurement sample; usinga light source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a side angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide method foranalyzing particles and/cells in a sample, comprising: receiving thesample into a cartridge device comprising a flow cell; using thecartridge device to mix the sample the sample with a reagent to form ameasurement sample; using the flow cell to form a sample flow of themeasurement sample; using a light source to emit an irradiation light;using the irradiation light to illuminate the sample flow; using acollecting lens to collect both a scattered light and a fluorescentlight from the particles and/or cells in the sample flow; and using one,two, or more detectors to detect a signal of the scattered light and asignal of the fluorescent light. In some embodiments, the scatteredlight comprises a forward scattered light, that is, a scattered lightwith a forward angle (e.g., a scattering angle less than about 25degrees). In other embodiments, the scattered light comprises a sidescattered light, that is, a scattered light with a side angle (e.g., ascattering angle more than about 25 degrees).

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the present disclosure are described indetail below with reference to the attached drawing figures.

FIG. 1A illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a device or device system asdisclosed herein, which comprises a sample supply unit, a detectionunit, and a signal analysis unit.

FIG. 1B illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a detection unit, whichcomprises a light source, a focusing module, a flow cell, a collectinglens, a receiving module, and a detector.

FIGS. 2A and 2B illustrate, in accordance with various embodiments ofthe disclosure, a non-limiting example of the detection unit, in which aflow cell is used to form the sample flow of the measurement sample.

FIG. 3A illustrates, in accordance with various embodiments of thedisclosure, that when blocking the irradiation light, the beam stopperalso blocks the scattered light with a scattering angle less than θ₁.

FIG. 3B illustrates, in accordance with various embodiments of thedisclosure, that the beam stopper can be a light obstruction barcomprising opaque material.

FIG. 4A illustrates, in accordance with various embodiments of thedisclosure, that the beam stopper can be positioned between the flowcell and the collecting lens.

FIG. 4B illustrates, in accordance with various embodiments of thedisclosure, that the beam stopper can be positioned behind thecollecting lens.

FIG. 5A illustrates, in accordance with various embodiments of thedisclosure, that a spherical lens has at least one curved surface thathas a spherical shape.

FIG. 5B illustrates, in accordance with various embodiments of thedisclosure, that an aspherical lens has at least one curved surface withan aspherical shape defined by an equation.

FIGS. 6A and 6B illustrate, in accordance with various embodiments ofthe disclosure, that the flow cell can be positioned at or close to thefocal point of the one lens (FIG. 6A) and away from the focal point ofanother lens (FIG. 6B).

FIGS. 6C and 6D illustrate, in accordance with various embodiments ofthe disclosure, that the diameter (d₁) of the elliptical beam spot alongthe sample flow's direction (i.e., the diameter along the y-axis) isnarrow and that the diameter (d₂) of the elliptical beam spotperpendicular to the sample flow's direction (i.e., the diameter alongthe x-axis) is wide.

FIG. 7A illustrates, in accordance with various embodiments of thedisclosure, a non-limiting example where a circular beam spot is used toirradiate the sample flow.

FIG. 7B illustrates, in accordance with various embodiments of thedisclosure, that when the beam spot and the sample flow have analignment deviation ΔX, the beam spot can no longer irradiate the sampleflow.

FIG. 7C illustrates, in accordance with various embodiments of thedisclosure, a non-limiting example where an elliptical beam spot is usedto irradiate the sample flow.

FIG. 7D illustrates, in accordance with various embodiments of thedisclosure, that with the same alignment deviation ΔX, the beam spot canstill irradiate the sample flow.

FIG. 8 illustrates, in accordance with various embodiments of thedisclosure, a zoom-in view of the detection unit from FIG. 2B.

FIGS. 9A and 9B illustrate, in accordance with various embodiments ofthe disclosure, another non-limiting example to obtain the ellipticalbeam spot, where the focusing module 903 comprises a condenser lens 909and one cylindrical lens 911.

FIG. 10A illustrates, in accordance with various embodiments of thedisclosure, a flow cell with sheath flow can be used, where the sampleflow is surrounded by a sheath flow in the flow cell.

FIG. 10B illustrates, in accordance with various embodiments of thedisclosure, a flow cell without sheath flow can be used, where there isno sheath flow surrounding the sample flow in the flow cell.

FIG. 11 illustrates, in accordance with various embodiments of thedisclosure, a non-limiting example of the light source, which comprisesa light-emitting component, an optical fiber, and a condenser lens.

FIG. 12 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a scatter plot of fluorescenceand scattered light, in which each dot represents one white blood cellbeing detected in the sample flow.

FIG. 13 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a histogram plot, in which thefluorescence intensities are plotted as the x-axis, and the numbers ofthe detected cells with the corresponding fluorescence intensities areplotted as the y-axis.

FIG. 14 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a scattered plot of fluorescenceand scattered light, in which each dot represents one red blood cell orplatelet being detected in the sample flow.

FIG. 15 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of a histogram plot, in which theintensities of the scattered light are plotted as the x-axis, and thenumbers of the detected cells with the corresponding intensities of thescattered light are plotted as the y-axis

FIGS. 16A and 16B illustrate, in accordance with various embodiments ofthe disclosure, another non-limiting example of the detection unit.

FIG. 16C illustrates, in accordance with various embodiments of thedisclosure, a zoom-in view of the light source, the focusing module andthe flow cell. In this non-limiting example, the optical axis of thespherical lens 1605 and the optical axis of the cylindrical lens 1606are coaxial, and they are further coaxial with the center axis 1616 ofthe light emitted from the light source.

FIG. 16D illustrates, in accordance with various embodiments of thedisclosure, a zoom-in view of the light source, the focusing module andthe flow cell. In this non-limiting example, the optical axis of thespherical lens 1605 and the optical axis of the cylindrical lens 1606are coaxial, but they are not coaxial with the center axis 1616 of thelight emitted from the light source.

FIG. 16E illustrates, in accordance with various embodiments of thedisclosure, a non-limiting example of a detection unit, in which the twolenses 1605 and 1606 are coaxial with each other, but not coaxial withthe center axis 1616 of the light emitted from the light source 1602.

FIG. 16F illustrates, in accordance with various embodiments of thedisclosure, a non-limiting example of a detection unit, in which thelens 1606 is coaxial with the center axis 1616 of the emitted light, butthe lens 1605 is not coaxial with the center axis 1616 of the emittedlight.

FIG. 16G illustrates, in accordance with various embodiments of thedisclosure, a zoom-in view of the light source, the focusing module andthe flow cell. In this non-limiting example, the flow cell 1601 istilted in a way that the surface 1621 of the flow cell is notperpendicular to the irradiation light 1617. For example, the angle θ₃between the surface 1621 and the center axis 1616 of the irradiationlight 1617 is not equal to 90 degrees.

FIG. 17 illustrates an example embodiment of a doublet lens that may beused with any of the embodiments disclosed herein.

FIGS. 18A and 18B illustrate, in accordance with various embodiments ofthe disclosure, a non-limiting example of the detection unit, in whichthe collection efficiency is improved with a doublet lens.

DETAILED DESCRIPTION

The following describes some non-limiting exemplary embodiments of theinvention with references to the accompanying drawings. The describedembodiments are merely a part rather than all of the embodiments of theinvention. All other embodiments obtained by a person of ordinary skillin the art based on the embodiments of the disclosure shall fall withinthe scope of the disclosure.

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. Tabelling, Introduction to Microfluidics reprint edition,Oxford University Press (2010); Hguyen et al., Fundamentals andApplications of Microfluidics 2^(nd) ed., Artech House Incorporated(2006); Berg et al., Microfluidics for Medical Applications, RoyalSociety of Chemistry (2014); Gomez et al., Biological Applications ofMicrofluidics 1^(st) ed., Wiley-Interscience (2008); and Colin et al.,Microfluidics 1^(st) ed., Wiley-ISTE (2010), provide one skilled in theart with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present disclosure. Other features and advantages of thedisclosure will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the disclosure.Indeed, the present disclosure is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. It should be understood that this disclosure is not limited tothe particular methodology, devices, systems, protocols, and reagents,etc., described herein and as such can vary. The definitions andterminology used herein are provided to aid in describing particularembodiments, and are not intended to limit the claims.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not. It will be understood by those withinthe art that, in general, terms used herein are generally intended as“open” terms (e.g., the term “including” should be interpreted as“including but not limited to,” the term “having” should be interpretedas “having at least,” the term “includes” should be interpreted as“includes but is not limited to,” etc.).

Unless stated otherwise, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of claims) can be construedto cover both the singular and the plural. The recitation of ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range.Unless otherwise indicated herein, each individual value is incorporatedinto the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the application and does not pose alimitation on the scope of the application otherwise claimed. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.” No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the application.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a light source configured to emit an irradiationlight for illuminating a sample flow, wherein the sample flow comprisesparticles and/or cells; a collecting lens configured to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aforward angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a flow cell configured to form a sample flow of ameasurement sample, wherein the measurement sample comprises particlesand/or cells; a light source configured to emit an irradiation light forilluminating the sample flow; a collecting lens configured to collectboth a scattered light with a forward angle and a fluorescent light fromthe particles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aforward angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a light source configured to emit an irradiationlight for illuminating a sample flow, wherein the sample flow comprisesparticles and/or cells; a collecting lens configured to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aside angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

Various embodiments of the present disclosure provide a device or devicesystem that comprises: a flow cell configured to form a sample flow of ameasurement sample, wherein the measurement sample comprises particlesand/or cells; a light source configured to emit an irradiation light forilluminating the sample flow; a collecting lens configured to collectboth a scattered light with a side angle and a fluorescent light fromthe particles and/or cells in the sample flow; and one, two, or moredetectors configured to detect a signal of the scattered light with aside angle and a signal of the fluorescent light. A device or devicesystem as disclosed herein can be used for analyzing particles and/orcells in a sample.

In various embodiments, a device or device system as described hereinfurther comprises a focusing module configured to focus the irradiationlight to form an elliptical beam spot on the sample flow. In variousembodiments, a device or device system as described herein furthercomprises a receiving module configured to split the scattered light andthe fluorescent light collected by the collecting lens into two separateoptical paths toward two separate detectors, respectively. In variousembodiments, a device or device system as described herein furthercomprises a doublet lens configured to focus the collected fluorescentlight. In various embodiments, a device or device system as describedherein further comprises a signal analysis unit configured to analyzethe signal of the scattered light and the signal of the fluorescentlight for analyzing the particles and/or cells.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a sample flow, comprising: using alight source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a forward angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a measurement sample, comprising:using a flow cell to form a sample flow of the measurement sample; usinga light source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a forward angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a forward angleand a signal of the fluorescent light. In some embodiments, two separatedetectors are used to detect the signal of the scattered light with aforward angle and the signal of the fluorescent light. In variousembodiments, the method further comprises using a focusing module tofocus the irradiation light to form an elliptical beam spot on thesample flow. In various embodiments, the method further comprises usinga receiving module to split the scattered light with a forward angle andthe fluorescent light collected by the collecting lens into two separateoptical paths toward two separate detectors, respectively. In variousembodiments, the method further comprises using a doublet lens to focusthe collected fluorescent light. In various embodiments, the methodfurther comprises using a signal analysis unit to analyze the signal ofthe scattered light with a forward angle and the signal of thefluorescent light for analyzing the particles and/or cells.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a sample flow, comprising: using alight source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a side angleand a signal of the fluorescent light.

Various embodiments of the present disclosure provide a method ofanalyzing particles and/or cells in a measurement sample, comprising:using a flow cell to form a sample flow of the measurement sample; usinga light source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a side angle and a fluorescent light from theparticles and/or cells in the sample flow; and using one, two, or moredetectors to detect a signal of the scattered light with a side angleand a signal of the fluorescent light. In some embodiments, two separatedetectors are used to detect the signal of the scattered light with aside angle and the signal of the fluorescent light. In variousembodiments, the method further comprises using a focusing module tofocus the irradiation light to form an elliptical beam spot on thesample flow. In various embodiments, the method further comprises usinga receiving module to split the scattered light with a side angle andthe fluorescent light collected by the collecting lens into two separateoptical paths toward two separate detectors, respectively.

In various embodiments, the method further comprises using a doubletlens to focus the collected fluorescent light. In various embodiments,the method further comprises using a signal analysis unit to analyze thesignal of the scattered light with a side angle and the signal of thefluorescent light for analyzing the particles and/or cells.

Various embodiments of the present disclosure provide method foranalyzing particles and/cells in a sample, comprising: receiving thesample into a cartridge device comprising a flow cell; using thecartridge device to mix the sample the sample with a reagent to form ameasurement sample; using the flow cell to form a sample flow of themeasurement sample; using a light source to emit an irradiation light;using the irradiation light to illuminate the sample flow; using acollecting lens to collect both a scattered light and a fluorescentlight from the particles and/or cells in the sample flow; and using one,two, or more detectors to detect a signal of the scattered light and asignal of the fluorescent light. In some embodiments, the scatteredlight comprises a forward scattered light, that is, a scattered lightwith a forward angle (e.g., a scattering angle less than about 25degrees). In other embodiments, the scattered light comprises a sidescattered light, that is, a scattered light with a side angle (e.g., ascattering angle more than about 25 degrees). In various embodiments,two separate detectors are used to detect the signal of the scatteredlight and the signal of the fluorescent light. In various embodiments,the method further comprises using a focusing module to focus theirradiation light to form an elliptical beam spot on the sample flow. Invarious embodiments, the method further comprises using a receivingmodule to split the scattered light and the fluorescent light collectedby the collecting lens into two separate optical paths toward twoseparate detectors, respectively. In various embodiments, the methodfurther comprises using a doublet lens to focus the collectedfluorescent light. In various embodiments, the method further comprisesusing a signal analysis unit to analyze the signal of the scatteredlight and the signal of the fluorescent light for analyzing theparticles and/or cells.

In various embodiments, the light source comprises a laser diode, alight-emitting diode (LED), a laser module, or a halogen lamp, or acombination thereof. In some embodiments, the light source comprises alaser diode and an optical fiber.

In various embodiments, the collecting lens comprises a spherical lens,an aspherical lens, or a doublet lens, or a combination thereof. In someembodiments, the collecting lens is a spherical lens. In someembodiments, the collecting lens is an aspherical lens. In someembodiments, the collecting lens is a doublet lens.

In various embodiments, a device or device system as disclosed hereincomprises two separate detectors: one is configured to detect the signalof the scattered light with a forward angle and the other is configuredto detect the signal of the fluorescent light. In various embodiments, adevice or device system as disclosed herein comprises two separatedetectors: one is configured to detect the signal of the scattered lightwith a side angle and the other is configured to detect the signal ofthe fluorescent light.

In various embodiments, a device or device system as disclosed hereincomprises a first detector configured to detect the signal of thescattered light with a forward angle, and a second detector configuredto detect the signal of the fluorescent light. In various embodiments, adevice or device system as disclosed herein comprises a first detectorconfigured to detect the signal of the scattered light with a sideangle, and a second detector configured to detect the signal of thefluorescent light. In accordance with the present disclosure, the terms“first” and “second” are used to identify different detectors and theydo not indicate any sequential relationship.

In various embodiments, the detector for the fluorescent light comprisesa photodiode, an avalanche photodiode, or a silicon photomultiplier, ora combination thereof.

In various embodiments, the collected scattered light includes a forwardscattered light with a scattering angle less than about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25degrees. In various embodiments, the detected forward scattered lightincludes a scattered light with a scattering angle less than about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 degrees.

In various embodiments, the collected scattered light includes a sidescattered light with a scattering angle more than about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees. In variousembodiments, the detected scattered light includes a side scatteredlight with a scattering angle more than about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, or 90 degrees.

In various embodiments, a device or device system as described hereinfurther comprises a focusing module.

In some embodiments, the focusing module is configured to focus theirradiation light to form an elliptical beam spot on the sample flow. Insome embodiments, the focusing module comprises a lens that is eithernot coaxial or not perpendicular with the central axis of irradiationlight.

In various embodiments, the elliptical beam spot has a width larger thanthe width of the flow cell. In various embodiments, the elliptical beamspot covers more than the whole width of the flow cell. In variousembodiments, the major axis (d₂) of the elliptical beam spot isperpendicular to the direction of the sample flow and the minor axis(d₁) of the elliptical beam spot is along the direction of the sampleflow. In various embodiments, the d₂:d₁ ratio is more than 1 or in therange of about 2-5, 5-10, 10-15, 15-20, or 20-25.

In various embodiments, the major axis (d₂) of the elliptical beam spotis larger than the width (d₃) of the flow cell. In various embodiments,the d₂:d₃ ratio is in the range of about 2-5, 5-10, 10-15, 15-20, or20-25.

In various embodiments, the elliptical beam spot on the flow cell has adiameter of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-99, or 99-100 μm in the direction parallel to thesample flow, and a diameter of about 40-100, 100-500, 500-1000,1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000,40004500, or 4500-5000 μm in the direction perpendicular to the sampleflow. In some embodiments, the elliptical beam spot on the flow cell hasa diameter of about 15-16, 16-20, 20-30, 30-40, or 40-50 μm in thedirection parallel to the sample flow, and a diameter of about 150-160,160-200, 200-500, 500-1000, 1000-1500, 1500-2000, or 2000-2500 μm in thedirection perpendicular to the sample flow.

In various embodiments, the sample flow formed in the flow cell has awidth of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, or 90-100 μm in the direction perpendicular to the sampleflow. In some embodiments, the sample flow formed in the flow cell has awidth of about 20-30, 30-40, or 40-50 μm in the direction perpendicularto the sample flow.

In various embodiments, the flow cell has a width of about 4-5, 5-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 μm inthe direction perpendicular to the sample flow. In some embodiments, theflow cell has a width of about 20-30, 30-40, or 4050 μm in the directionperpendicular to the sample flow. In various embodiments, the flow cellhas a width in the range of about 1-10, 10-40, 40-100, or 100-200 μm;and a depth in the range of about 1-10, 10-40, 40-100, or 100-200 μm. Invarious embodiments, the flow cell has a length in the range of about1-10, 10-100, 100-1,000, 1,000-5,000 μm, or 5,000-10,000 μm.

In some embodiments, the flow cell is configured to form the sample flowwithout a sheath flow. In other embodiments, the flow cell is configuredto form the sample flow with a sheath flow. In some embodiments, thesample flow has no sheath flow. In other embodiments, the sample flow issurrounded by a sheath flow.

In various embodiments, the flow cell comprises a surface that isilluminated by the irradiation light, and the surface is positioned tobe not perpendicular with the central axis of the irradiation light. Insome embodiments, the angle between the surface and the central axis ofthe irradiation light can be about 45-50, 50-55, 55-60, 60-65, 65-70,70-75, 75-80, 8085, 85-89, or 89-89.9 degrees.

In various embodiments, the flow cell is part of a cartridge deviceconfigured to be placed into a reader instrument for analysis, andwherein the reader instrument comprises a light source, a collectinglens, detectors, and a signal analysis unit. In various embodiments, thecartridge device comprises a surface that is illuminated by theirradiation light, and the surface is positioned to be not perpendicularwith the central axis of the irradiation light. In some embodiments, theangle between the surface and the central axis of the irradiation lightcan be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85,85-89, or 89-89.9 degrees.

In various embodiments, the flow cell or the cartridge device that hoststhe flow cell is positioned or titled in such an orientation that areflective surface of the flow cell or the cartridge device is notperpendicular to the central axis of the irradiation light and directsthe reflected light away from the light source. In certain embodiments,the angle between such a reflective surface and the central axis of theirradiation light can be about 45-50, 50-55, 5560, 60-65, 65-70, 70-75,75-80, 80-85, 85-89, or 89-89.9 degrees.

In some other embodiments, a lens in the focusing module is positionedor titled in such an orientation that a reflective surface of the lensis not perpendicular to the central axis of the irradiation light anddirects the reflected light away from the light source. In certainembodiments, the angle between such a reflective surface and the centralaxis of the irradiation light can be about 45-50, 50-55, 55-60, 60-65,65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees.

In various embodiments, the cartridge device is configured to mix asample with a reagent to form the measurement sample and to form asample flow of the measurement sample in the flow cell. In variousembodiments, a method as described herein further comprises using thecartridge device to mix a sample with a reagent to form the measurementsample and to form a sample flow of the measurement sample in the flowcell. In various embodiments, the reagent comprises a fluorescentlabeling compound, an osmolality-adjusting compound, a spheringcompound, or a lysing compound, or a combination thereof. Non-limitingexamples of the reagent can be found in the present disclosure and U.S.patent application Ser. No. 15/819,416, which are incorporated herein byreference in their entirety as if fully set forth.

In various embodiments, the reagent comprises a fluorescent labelingcompound. In various embodiments, the fluorescent labeling compoundcomprises an antibody conjugated with a fluorophore, an antibodyconjugated with a fluorescent particle, or a fluorescent dye, orcombination thereof. In various embodiments, the reagent comprises afluorescent dye.

In various embodiments, a device or device system as described hereinfurther comprises a receiving module.

In various embodiments, the receiving module is configured to split thescattered light with a forward angle and the fluorescent light collectedby the collecting lens into two separate optical paths toward twoseparate detectors, respectively. In various embodiments, the receivingmodule is configured to split the scattered light with a side angle andthe fluorescent light collected by the collecting lens into two separateoptical paths toward two separate detectors, respectively. In variousembodiments, the receiving module comprises a beam splitter, a dichroicmirror, a prism, or a diffractive grating, or a combination thereof. Insome embodiments, the receiving module comprise a doublet lensconfigured to focus the collected fluorescent light.

In various embodiments, a device or device system as described hereinfurther comprises a doublet lens configured to focus the collectedfluorescent light.

In various embodiments, a device or device system as described hereinfurther comprises a signal analysis unit configured to analyze thesignal of the scattered light with a forward angle and the signal of thefluorescent light for analyzing the particles and/or cells.

In various embodiments, the measurement sample comprises blood cells. Invarious embodiments, the sample comprises blood cells.

In various embodiments, analyzing the particles and/or cells comprisesanalyzing blood cells. In various embodiments, analyzing blood cellscomprises one or more of: measuring the number and/or percentage ofwhite blood cells, identifying white blood cells into subtypes (e.g.,lymphocytes, monocytes, neutrophils, eosinophils, and basophils),measuring the number and/or percentage of a subtype of white bloodcells, measuring the number and/or percentage of red blood cells, andmeasuring the number and/or percentage of platelets. In someembodiments, analyzing blood cells comprises measuring the number of redblood cells and the number of platelets.

In various embodiments, a device or device system as disclosed hereincomprises a sample supply unit 101, a detection unit 102, and a signalanalysis unit 103, as shown in FIG. 1A. The sample supply unit providesto the detection unit a measurement sample containing cells, orparticles, or both. In various embodiments, the detection unit comprisesa light source 104, a focusing module 105, a flow cell 106, a collectinglens 107, a receiving module 108, and a detector 109, as shown in FIG.1B. The sample supply unit provides a measurement sample to the flowcell of the detection unit. The measurement sample is passed through theflow cell to form a sample flow. Light emitted from the light source isused to irradiate the sample flow and light signals from the sample flow(e.g., scattered light with a forward angle, fluorescence, or both) aremeasured by the detector. Before irradiating the sample flow, the lightemitted from the light source is shaped into a desired beam spot by thefocusing module. Before being measured by the detector, the lightsignals from the sample flow are collected by the collecting lens anddirected by the receiving module towards the detector. The signalanalysis unit analyzes the light signals measured by the detector toobtain desired results (e.g., the number of cells, characteristics ofindividual cells, and the distribution and characterization of cellpopulations in the measurement sample). The receiving module can be usedfor various other functions: to separate the collected light intomultiple optical paths, to filter out certain wavelengths from thecollected light, to focus the collected light into a target spot ofparticular size or shape, or to perform other light processingfunctionalities. In various embodiments, one, two, or more detectors canbe used for measuring one, two, or more types of light signals from thesample flow.

FIG. 2A (side view) and FIG. 2B (top view) show a non-limiting exampleof the detection unit, in which a flow cell 201 is used to form thesample flow of the measurement sample. A light source 202 emits anirradiation light to irradiate the sample flow. A focusing module 203focuses and shapes the irradiation light into a particular beam shape onthe sample flow. A collecting lens 204 collects both scattered lightwith a forward angle and fluorescence from the sample flow illuminatedwith the irradiation light. A receiving module 205 separates thecollected light into two optical paths. Two detectors, detector 206 anddetector 207, detect and measure the collected light on the two opticalpaths. A beam stopper 208 can be positioned in the optical path betweenthe flow cell and one of the detectors and blocks the irradiation lightthat passes through the flow cell.

In this non-limiting example, the focusing module 203 comprises acondenser lens 209, and two cylindrical lenses 210 and 211. Theirradiation light emitted from the source 201 is collimated by thecondenser lens 209 and forms parallel light with either a circular beamshape or an elliptical beam shape. The irradiation light further passesthrough the cylindrical lenses 210 and 211. The two cylindrical lensesare positioned in such an orientation that the cylinder axis of the lens210 and the cylinder axis of the lens 211 are perpendicular to eachother. The flow cell 201 is positioned at or close to the focal point ofthe lens 211 and away from the focal point of the lens 210, where theirradiation light is focused and shaped into a light beam with anelliptical shape on the sample flow.

The collecting lens 204 is a condenser lens and the flow cell 201 can bepositioned at or close to its focal point. The signal from themeasurement sample is collected and condensed by the collecting lens 204before entering the receiving module 205. The collected signal includesbut is not limited to scattered light with a forward angle andfluorescence from the sample flow. The signal from the measurementsample is collimated with the collecting lens 204 into parallel light.Furthermore, a beam stopper 208 can be positioned behind the collectinglens 204 to block the irradiation light from entering the receivingmodule.

The receiving module 205 comprises a dichroic mirror 212, which istilted at a 45-degree angle relative to the collected parallel light.The dichroic mirror 212 is a long-pass dichroic mirror, which reflectsthe light with a wavelength below a designated threshold. The scatteredlight from the sample flow has a wavelength that is the same or close tothe irradiation light. The fluorescence from the sample flow has awavelength spectrum including wavelengths longer than the irradiationlight. By choosing a dichroic mirror with a threshold wavelength that islonger than the irradiation light but shorter than the desiredfluorescence, it separates the scattered light and the fluorescence intotwo optical paths. Alternatively, the receiving module can use otheroptical configurations (e.g., a beam splitter, a combination of a beamsplitter and optical filters, a prism, a diffractive grating, etc.) toseparate the scattered light and the fluorescence. In this example, acondenser lens 213 is positioned in front of the detector 206, andcondenses the light passing through the dichroic mirror on the detector.The light reflected by the dichroic mirror is received in the detector207. An aperture 214 made with an opaque material and having atransparent opening in the center can be positioned in front of thedetector 207. The aperture 214 blocks the light outside the transparentopening from entering the detector 207.

The collecting lens 204 in the detection unit is used to collect boththe scattered light with a forward angle and the fluorescence from thesample flow. In some embodiments, the collected scattered lightcomprises light with a scattering angle less than about 20 degrees. Insome embodiments, the collected scattered light comprises light with ascattering angle less than about 15 degrees. In some embodiments, thecollected scattered light comprises light with a scattering angle lessthan about 10 degrees. In some embodiments, the collected scatteredlight comprises light with a scattering angle less than about 5 degrees.In some embodiments, the collected scattered light comprises light witha scattering angle less than about 4 degrees. In some embodiments, thecollected scattered light comprises light with a scattering angle lessthan about 3 degrees. In some embodiments, the collected scattered lightcomprises light from elastic scattering. In some embodiments, thecollected scattered light comprises light from non-elastic scattering.In various embodiments, the collecting lens is a spherical lens, anaspherical lens, or a doublet lens, or a combination thereof.

In traditional analyzers, such as the one described in U.S. Pat. No.7,580,120, the scattered light with a forward angle and the fluorescenceare not collected in the same collecting lens; instead, the scatteredlight with a forward angle is collected in one collecting lens and thefluorescence is collected in another collecting lens. The lenscollecting the fluorescence is positioned perpendicular to the directionof the irradiation light, and thus collects only scattered light with aside angle, which usually having a scattering angle more than about 70degrees. This configuration requires the flow cell to have at least twooptical transparent surfaces, one for the collection of the scatteredlight with a forward angle and the other for the collection of thefluorescence signals.

On the contrary, by collecting the fluorescence and the scattered lightwith a forward angle in the same collecting lens, one opticaltransparent surface is sufficient for the detection of both signals. Inlow-cost flow cells, such as those built with plastic injection molding,it is more cost-effective to have one optical transparent surface thantwo optical transparent surfaces.

The intensity of the fluorescence and the scattered light could besignificantly lower than the intensity of the irradiation light. Inorder to detect signals with satisfactory signal-to-noise ratio (SNR),therefore, it is desirable to remove the irradiation light from thecollected light. In the non-limiting example of FIG. 2A and FIG. 2B, abeam stopper 208 is used to block the irradiation light behind the flowcell. When blocking the irradiation light 302, as shown in FIG. 3A, thebeam stopper 303 also blocks the scattered light 305 with a scatteringangle less than θ₁. The value θ₁ is adjustable by the size and shape ofthe beam stopper 303, as well as the distance from the beam stopper 303to the sample flow 301. In a non-limiting example as shown in FIG. 3B,the beam stopper 308 can be a light obstruction bar comprising opaquematerial. The size of bar is designated to be larger than the beam spotof the irradiation light 309 at the position of the beam stopper. Insome embodiments, the beam stopper has a surface that minimizes thereflection of the irradiation light to reduce stray light in thedetection unit. Examples of the surface include but are not limited tosurfaces made with light absorptive materials (e.g., black painting, andanodized aluminum), etc.

The beam stopper is positioned in the optical path between the flow celland one of the detectors to block the irradiation light. In anon-limiting example of FIG. 4A, the beam stopper 403 is positionedbetween the flow cell 405 and the collecting lens 404. In anothernon-limiting example of FIG. 4B, the beam stopper 403 is positionedbehind the collecting lens 405.

In some embodiments, it is advantageous to position the beam stopperbehind the collecting lens. To collect the fluorescence from the sampleflow, it is preferred that the collecting lens is close in distance tothe flow cell, so as to enlarge the collection angle of thefluorescence. If the beam stopper is positioned between the flow celland the collecting lens, then the distance from the sample flow to thebeam stopper is limited, and a given size of the beam stopper may blockthe scattered light with a significantly large θ₁. By positing the beamstopper behind the collecting lens, it increases the distance from thesample flow to the beam stopper, without sacrificing the distance fromthe sample flow to the collecting lens. In this way, the angle θ₁ couldbe reduced for a given size of the beam stopper. In some embodiments,the collecting lens itself may have a thickness in the range of 5-15 mm.This lens thickness could help to add considerable distance between thesample flow and the beam stopper.

In some embodiments, a spherical lens is used as the collecting lens tocollect the fluorescence and the scattered light with a forward anglefrom the sample flow. The spherical lens has at least one curved surfacethat has a spherical shape, as shown in FIG. 5A. In some embodiments, anaspherical lens is used as the collecting lens to collect thefluorescence and the scattered light with a forward angle. An asphericallens is advantageous to increase the collection efficiency of thefluorescence from the sample flow. The aspherical lens, as shown in FIG.5B, has at least one curved surface with an aspherical shape defined bythe following equation.

$X = {\frac{C_{0} \cdot Y^{2}}{1 + \sqrt{1 - {\left( {K + 1} \right) \cdot C_{0}^{2} \cdot Y^{2}}}} + {\sum\limits_{i = 1}^{n}\;{C_{i}Y^{i}}}}$

In the above equation: X represents position in the optical axisdirection; Y represents distance from the lens center in the directionin which the optical axis advances; K represents shape coefficient; C₀represents coefficient representing the curvature of the based surface(spherical surface basis of the aspherical surface); C representsaspherical surface coefficient; and i represents an integer (1 to n).

In the detection unit of FIG. 2A and FIG. 2B, an elliptical beam spot isused to irradiate the sample flow in the flow cell. This elliptical beamspot is achieved by using the two cylindrical lenses 210 and 211. Thetwo cylindrical lenses are positioned in such an orientation that theircylinder axes are perpendicular to each other. The flow cell ispositioned at or close to the focal point of the lens 211, as shown inFIG. 6A. Therefore, the diameter (d₁) of the elliptical beam spot alongthe sample flow's direction is narrow, i.e., the diameter along they-axis as shown in FIG. 6C and FIG. 6D. Meanwhile, the flow cell ispositioned away from the focal point of the lens 210, as shown in FIG.6B. Therefore, the diameter (d₂) of the elliptical beam spotperpendicular to the sample flow's direction is wide, i.e., the diameteralong the x-axis as shown in FIG. 6C and FIG. 6D. The aspect ratio R ofthe elliptical beam spot is defined as follows.R=d ₂ /d ₁

An elliptical beam spot has an aspect ratio R>1. When R=1, the beam spotbecomes a circular shape. The sample flow width irradiated under thebeam spot, as shown in FIG. 6D, is denoted as d₃. One advantage of usingan elliptical beam spot is that the two diameters, d₁ and d₂, could beoptimized separately. In various embodiments, d₁ and d₂ can be adjustedin a device or device system as described herein by adjusting thefocusing module configuration, for example, adjusting the positions ofthe light source, the two cylindrical lenses 210 and 211, and the flowcell, or changing these components.

For example, the diameter d₁ can be optimized for measuring cells ofdifferent sizes. To fully irradiate the cells with the beam spot, thediameter d₁ has to be larger than the diameter of the target cells. Toreduce the background being irradiated by the beam spot, however, thediameter d₁ should be as small as possible. Therefore, the diameter d₁is often chosen to be close to or slightly larger than the diameters ofthe target cells. This is helpful to increase the amplitude of thefluorescence from the measurement sample and to improve thesignal-to-noise ratio of the fluorescence from the target cells.

In some embodiments, a d₁ of about 4-7 μm is used for measuring cellswith a diameter of about 1-3 μm. In some embodiments, a d₁ of about 7-10μm is used for measuring cells with a diameter of about 1-6 μm. In someembodiments, a d₁ of about 10-20 μm is used for measuring cells with adiameter of about 1-9 μm. In some embodiments, a d₁ of about 20-30 μm isused for measuring cells with a diameter of about 1-19 μm. In someembodiments, a d₁ of about 30-50 μm is used for measuring cells with adiameter of about 1-29 μm. In some embodiments, a d₁ of about 50-80 μmis used for measuring cells with a diameter of about 1-49 μm. In someembodiments, a d₁ of about 80-99 μm is used for measuring cells with adiameter of about 1-79 μm.

For another example, the diameter d₂ can be optimized for alignmentbetween the sample flow and the irradiation light in the beam spot. Sometraditional analyzers use a circular beam spot to irradiate the sampleflow, such as the one disclosed in US Patent Application Publication No.2015/0309049 A1. A circular beam spot has an aspect ratio of R=1 (i.e.,d₂=d₁). After d₁ is chosen for cell sizes, the circular beam spot limitsthe option of d₂. FIG. 7A shows a non-limiting example where a circularbeam spot is used to irradiate the sample flow. When the beam spot andthe sample flow have an alignment deviation ΔX, as shown in FIG. 7B, thebeam spot is no longer able to irradiate the sample flow. For example,when the beam spot has a d₁ of 20 μm, the circular beam spot limits d₂to be equal to d₁. For a sample flow width d₃ of 20 μm, an alignmentdeviation of 20 μm would cause the beam spot not to irradiate the sampleflow.

On the contrary, in an elliptical beam spot, the two diameters, d₁ andd₂, could be optimized separately. The elliptical beam spot has anaspect ratio of R>1 (i.e., d₂>d₁). FIG. 7C shows a non-limiting examplewhere an elliptical beam spot is used to irradiate the sample flow. Withthe same amount of alignment deviation ΔX, as shown in FIG. 7D, the beamspot can still irradiate the sample flow. For example, when the beamspot has a d₁ of 20 μm, the elliptical beam spot can still have a dlarger than e.g., 200 μm. For a sample flow width d of 20 μm, the beamspot can still irradiate the sample flow even with an alignmentdeviation of 20 μm. The tolerance of alignment deviation between theflow cell and the beam spot is particularly important for applicationswhere the flow cell is replaceable or disposable after measurement.Furthermore, an irradiation light with a Gaussian beam profile is oftenused in cytometer analysis, and a wide d₂ also helps to improve theirradiation light's uniformity along the sample flow width.

In some embodiments, a d₂ of about 40-500 μm is used for irradiating aflow cell with a sample flow width of about 4-10 μm. In someembodiments, a d₂ of about 100-1,000 μm is used for irradiating a flowcell with a sample flow width of about 10-20 μm. In some embodiments, ad₂ of about 200-1,500 μm is used for irradiating a flow cell with asample flow width of about 20-30 μm. In some embodiments, a d₂ of about300-2,000 μm is used for irradiating a flow cell with a sample flowwidth of about 30-40 μm. In some embodiments, a d₂ of about 400-2,500 μmis used for irradiating a flow cell with a sample flow width of about40-50 μm. In some embodiments, a d₂ of about 500-5,000 μm is used forirradiating a flow cell with a sample flow width of about 50-100 μm.

In the example of FIG. 2A and FIG. 2B, the aperture 214 is used to blocklight outside the transparent opening of the aperture from entering thedetector 207. By positioning the transparent opening of the aperture 214at the center the detector 207 and by adjusting the size of the opening,the aperture can be used to block scattered light with a scatteringangle more than a threshold angle θ₂, as shown in FIG. 8, which is azoom-in view of the detection unit from FIG. 2B.

In the example of FIG. 2A and FIG. 2B, the elliptical beam spot isobtained by focusing the light emitted from the light source with afocusing module 203 that comprises two cylindrical lenses 210 and 211.In other embodiments, the elliptical beam spot can be obtained by usingother focusing module configurations, which include but are not limitedto modules with only one cylindrical lens, modules with no cylindricallens, modules with an anamorphic prism pair (e.g., U.S. Pat. No.5,596,456, which is incorporated herein by reference in its entirety asif fully set forth), modules with a diffraction grating component, andmodules with other beam-shaping optics (e.g., U.S. Pat. No. 6,975,458,which is incorporated herein by reference in its entirety as if fullyset forth), etc.

FIG. 9A (side view) and FIG. 9B (top view) shows another non-limitingexample to obtain the elliptical beam spot, where the focusing module903 comprises a condenser lens 909 and a cylindrical lens 911. The lightemitted from the light source 902 is collimated into parallel light withthe condenser lens 909, and further passes through the cylindrical lens911. Along the cylinder axis of lens 911, the light beam is focused bythe curved surface of the cylindrical lens (i.e., focused along they-axis here). Perpendicular to the cylinder axis of lens 911, the lightbeam is not focused and remains parallel. By positioning the flow cellat or close to the focal point of the cylindrical lens 901, theirradiation light forms an elliptical beam spot on the sample flow. Invarious embodiments, d₁ and d₂ can be adjusted in a device or devicesystem as described herein by adjusting the focusing moduleconfiguration, for example, adjusting the positions of the light source,the condenser lens 909, the cylindrical lens 911, and the flow cell, orchanging these components.

In this example, a collecting lens 904 is used to collect both thefluorescence and the scattered light with a forward angle from thesample flow. A beam stopper 908 is positioned behind the collecting lensto block the irradiation light from entering the receiving module 905.The receiving module 905 comprises a dichroic mirror 912, whichseparates the scattered light with a forward angle and the fluorescenceinto two optical paths. A condenser lens 913 condenses the fluorescencepassing through the dichroic mirror into the detector 906. An aperture914 in front of the detector 907 limits the scattered light receivableat the detector 907.

The detection unit can use any types of flow cell design, including butnot limited to a flow cell with sheath flow, a sheathless flow cell,etc. In some embodiments, a flow cell with sheath flow can be used,where the sample flow is surrounded by a sheath flow in the flow cell,as shown in FIG. 10A. The sheath flow 1002 focuses the sample flow 1003into a stream that has a width smaller than the inner width of the flowcell 1001 in the direction perpendicular to the sample flow 1003. Insome embodiments, a flow cell 1001 without sheath flow 1002 can be used,as shown in FIG. 10B. In this sheathless flow cell design, the sampleflow 1003 is confined by the physical geometry of the flow cell, and hasa width that is equal to the inner width of the flow cell 1001 in thedirection perpendicular to the sample flow 1003. In certain embodiments,the detection unit uses a sheathless flow cell. Examples of thesheathless flow cell include but are not limited to those disclosed inU.S. Patent Application No. 62/497,075, U.S. patent application Ser. No.15/803,133, and U.S. patent application Ser. No. 15/819,416, which areincorporated herein by reference in their entirety as if fully setforth.

The detection unit can use any types of light source to provide theirradiation light illuminating the sample flow, including but notlimited to a laser module, a laser diode, a LED device, a halogen lamp,etc. In some embodiments, as shown in FIG. 11, the light sourcecomprises a light-emitting component 1101, an optical fiber 1103, and acondenser lens 1102. The light-emitting component emits light, which isfocused by the condenser lens into one end of the optical fiber. Thelight exiting from the other end of the fiber is used to irradiate thesample flow. In certain embodiments, the optical fiber is a single-modefiber. It can be advantageous to use a single-mode fiber. For example,when a multi-mode light enters the single-mode fiber, some components ofthe light are removed by the fiber and the light existing from theoptical fiber becomes single-mode (e.g., the fundamental Gaussian mode).In certain embodiments, the light-emitting component is a laser diode, aLED device, or a halogen lamp.

The detection unit of the flow cytometer can use any types of lightdetectors to measure the fluorescence and the scattered light signals,including but not limited to bipolar phototransistor, photosensitivefield-effect transistor, photomultiplier tubes (PMT), avalanchephotodiode (APD), photodiode, CCD device, CMOS device, and siliconphotomultipliers (SiPM), etc. In some embodiments, the detection unitmeasures the intensity of the light signal. In some embodiments, thedetection unit measures the time duration of the light signal. In someembodiments, the detection unit measures the space distribution of thelight signal. In some embodiments, the detection unit measures the imageinformation of the light signal.

In various embodiments, a signal analysis unit is used to analyzesignals measured by detectors in the detection unit. In someembodiments, the signal analysis unit analyzes the signal of thescattered light and the signal of the fluorescent light from thedetection unit for measurement of particles and/or cells in the sampleflow.

In various embodiment, the flow cell is part of a cartridge device.Non-limiting examples of a cartridge device with a flow cell are shownin U.S. patent application Ser. No. 15/803,133 and U.S. patentapplication Ser. No. 15/819,416, which are incorporated herein byreference in their entirety as if fully set forth. In variousembodiment, the cartridge device is placed in an analyzer devicecomprising a light source, a collecting lens and a detector to performmeasurement of particles and/or cells in a sample flow in the flow cell.In various embodiment, the cartridge device is removed from the analyzerdevice after the measurement is completed. In some embodiments, thecartridge device receives a sample with particles and/or cells andfurther prepares a measurement sample from the sample with particlesand/or cells and a reagent, and then provides the measurement sample tothe flow cell to form a sample flow for the measurements.

A device or device system as described herein can be used for analyzingany types of samples containing cells. It can also be used for analyzingany types of samples containing particles, including but not limited toliquid droplets, molecules (e.g., nucleic acid molecules, proteinmolecules, etc.), viruses, beads, nanoparticles, etc. Its sample supplyunit provides to its detection unit a measurement sample containingcells, particles, or both. Its detection unit detects various signalsfrom the cells, particles, or both in the measurement sample. Itsanalysis unit analyzes the detected signals (e.g., scattered light witha forward angle, fluorescence, or both) to obtain information of themeasurement sample (e.g., cell count, intrinsic fluorescence ofindividual cells, fluorescence of individual cells labeled withfluorophore, etc.). Based on the detected signals, the analysis unit canfurther obtain additional information of the measurement sample, forexample, classifying cells into different types, characterizingindividual cells, characterizing cell populations in the measurementsample, etc.

In some embodiments, a device or device system as described herein isused for analyzing cells in blood samples (e.g., blood samples fromhuman or other species such as canine, feline, equine, bovine, ferret,gerbil, rabbit, pig, mini pig, and guinea pig, etc.). As a non-limitingexample, the device or device system can be used to analyze human bloodsamples, so as to detect and classify the cells in a human blood sampleinto three major types including white blood cells, red blood cells andplatelet cells. The device or device system can further be used toclassify white blood cells into five major subtypes including thelymphocytes, monocytes, neutrophils, eosinophils, and basophils. Thedevice or device system can further be used to detect the existence andlevel of antigen expressions on cells, and use the antigen expressionlevels to classify cells into different types. For a non-limitingexample, the device or device system can be used to classify lymphocytecells into T-Cells, NK-Cells, CD4⁺ cells, CD8⁺ cells, etc. The device ordevice system can be used to further detect and classify other cells inhuman blood samples, for example, those cells described in US PatentApplication Publication No. 2014/0170680 A1, which is incorporatedherein by reference in its entirety as if fully set forth.

For any specific type of biological samples for measurement, the cellsin the sample have known size ranges. Therefore, the size of the sampleflow in the flow cell and the diameters of the elliptical beam spot canbe optimized accordingly in the detection unit. For example, cells in ahuman blood sample can be analyzed. Human blood cells have known sizeranges, for example, about 1-3 μm in diameter for platelet cells, about6-8 μm in diameter for red blood cells, and about 7-15 μm in diameterfor white blood cells. Accordingly, the detection unit can use a sampleflow having d₃ of about 20-50 μm, and an elliptical beam spot having d₁of about 16-50 μm and d₂ of about 160-2,500 μm.

In some embodiments, a device or device system as described herein isused to analyze the white blood cells in a blood sample. In someembodiments, the sample supply unit prepares a measurement sample bymixing the blood sample with a staining reagent containing at least afluorescent labelling compound, which includes but is not limited tofluorophore-conjugated antibodies, fluorescent-particles-conjugatedantibodies, and fluorescent dyes, etc. The fluorescent labellingcompound labels the white blood cells with high affinity.

In some embodiments, the sample supply unit prepares a measurementsample by mixing the blood sample with a staining reagent containing atleast a fluorescent dye. This fluorescent dye can be a nucleic acid dye.Examples of the fluorescent dye include but are not limited to propidiumiodide, ethidium bromide, DAPI, Hoechst dyes, Acridine Orange, 7-AAD,LDS 751, TOTO families of dyes, TO-PRO families of dyes, SYTO family ofdyes, Thiazole Orange, Basic Orange 21, Auramine-O, and the dyecompounds disclosed in U.S. Pat. No. 6,004,816, etc., which isincorporated herein by reference in its entirety as if fully set forth.The fluorescent dye labels the nucleic acids of the white blood cellswith high affinity.

The prepared measurement sample is supplied to the flow cell of thedetection unit to form a sample flow. The sample flow is illuminatedwith an irradiation light, and the signals (e.g., fluorescence andscattered light with the forward angle) from the sample flow aremeasured by the two detectors in the detection unit. The analysis unitanalyzes the detected signals (e.g., intensities of the fluorescence andscattered light) to obtain the information of the measurement sample,which includes but is not limited to one or more of the followingparameters: the total count of the white blood cells, and the counts andpercentages of different subtypes of the white blood cell (e.g.,lymphocytes, monocytes, neutrophils, eosinophils and basophils, etc.).

In some embodiments, the staining reagent further comprises a lysingcompound, which lyses red blood cells in the blood sample. Because theconcentration of red blood cell is usually higher than the concentrationof white blood cells, it helps to improve the detection of the signalsfrom white blood cells in the sample flow. Examples of the lysingcompound include but are not limited to ammonium salts, quaternaryammonium salts, pyridinium salts, hydroxylamine salts, nonionicsurfactants, ionic surfactants, dodecyl sodium sulfate (SDS), sodiumlauryl sulfate (SLS), and their combinations, and any other knownerythrocyte lysing compounds.

In a non-limiting example, the fluorescent dye in the staining reagentis Thiazole Orange. A dichroic mirror has a long-pass thresholdwavelength of 560 nm is used to separate the fluorescence signal fromthe collected light. An obstruction bar is used as the beam stopper toblock the irradiation light. The obstruction bar has a bar width thatblocks the scattered light with a scattering angle θ₁ less than about 4degrees. An aperture having a transparent opening is used in front ofthe detector that receives the light comprising the scattered light, andthe aperture blocks the scattered light with a scattering angle θ₂ morethan about 12 degrees from entering the detector. The analysis unit usesthe detected signals of fluorescence and scattered light to produce ascatter plot. In one non-limiting example of the scatter plot, as shownin FIG. 12, each dot represents one white blood cell being detected inthe sample flow. The analysis unit enumerates the total number of dotsin the scatter plot to determine the total count of the white bloodcells in the blood sample. Furthermore, the dots in the scatter plotfall into distinguished clusters. The analysis unit enumerates thenumber of dots in each cluster to determine the counts and percentagesof different subtypes of the white blood cell including lymphocytes,monocytes, neutrophils, and eosinophils.

In another non-limiting example, the fluorescent dye in the stainingreagent is Acridine Orange. A dichroic mirror has a long-pass thresholdwavelength of 610 nm is used to separate the fluorescence signal fromthe collected light. The analysis unit uses the detected signals offluorescence to produce a histogram plot. In one non-limiting example ofthe histogram plot, as shown in FIG. 13, the fluorescence intensitiesare plotted as the x-axis, whereas the numbers of the detected cellswith the corresponding fluorescence intensities are plotted as they-axis. The histogram indicates three distinguished peaks. The peak withthe low fluorescence intensities corresponds to lymphocyte cells; thepeak with the middle fluorescence intensities corresponds to monocytecells; and the peak with high fluorescence intensities correspondsgranulocyte cells, which include the neutrophil cells, eosinophil cellsand basophil cells. The analysis unit enumerates the number of cells inall the peaks to determine the total count of the white blood cells, andfurther enumerates the number of cells in each peak to determine thecounts and percentages of lymphocyte cells, monocyte cells andgranulocyte cells.

In some embodiments, a device or device system as described herein isused to analyze the red blood cells and platelets in a blood sample. Insome embodiments, the sample supply unit prepares a measurement sampleby mixing the blood sample with a staining reagent containing at least afluorescent labelling compound, which includes but is not limited tofluorophore-conjugated antibodies, fluorescent-particle-conjugatedantibodies, and fluorescent dyes, etc. The fluorescent labellingcompound labels the red blood cells and platelets with high affinity.

In some embodiments, the sample supply unit prepares a measurementsample by mixing the blood sample with a staining reagent containing atleast a fluorescent dye. This fluorescent dye can be a nucleic acid dye.Examples of the fluorescent dye include but are not limited to propidiumiodide, ethidium bromide, DAPI, Hoechst dyes, Acridine Orange, 7-AAD,LDS 751, TOTO families of dyes, TO-PRO families of dyes, SYTO family ofdyes, Thiazole Orange, Basic Orange 21, Auramine-O, and the dyecompounds disclosed in U.S. Pat. No. 6,004,816, etc., which isincorporated herein by reference in its entirety as if fully set forth.The fluorescent dye labels the nucleic acids in the red blood cells andplatelets with high affinity.

The prepared measurement sample is supplied to the flow cell of thedetection unit to form a sample flow. The sample flow is illuminatedwith an irradiation light, and the signals (e.g., fluorescence andscattered light with the forward angle) from the sample flow aremeasured by the two detectors in the detection unit. The analysis unitanalyzes the detected signals (e.g., intensities of the fluorescence andscattered light) to obtain the information of the measurement sample,which includes but is not limited to one or more of the followingparameters: the total count of the red blood cells, the total count ofthe platelets, the sizes of individual red blood cells, the sizedistribution of the red blood cell population, the sizes of individualplatelets, and the size distribution of the platelet population, thecount of reticulocyte cells, and the count of immature platelet cells,etc.

In some embodiments, the staining reagent further comprises a spheringcompound. The sphering compound is used to transform the red blood cellsin the prepared sample from a disk shape into a spherical shape. With aspherical shape, the intensities of the scattered light from individualred blood cells become independent of the cells' orientation in the flowcell. Examples of the sphering compound include but are not limited tosurfactants such as sodium dodecyl sulfate (SDS) and sodium laurylsulfate (SLS), etc.

In a non-limiting example, the fluorescent dye used in the stainingreagent is Acridine Orange. A dichroic mirror has a long-pass thresholdwavelength of 590 nm is used to separate the fluorescence signal fromthe collected light. An obstruction bar is used as the beam stopper toblock the irradiation light. The obstruction bar has a bar width thatblocks the scattered light with a scattering angle θ₁ less than about 1degree. An aperture having a transparent opening is used in front of thedetector that receives the light comprising the scattered light, and theaperture blocks the scattered light with a scattering angle θ₂ more thanabout 5 degrees from entering the detector. The analysis unit uses thedetected signals of fluorescence and scattered light to produce ascatter plot. In one non-limiting example of the scattered plot, asshown in FIG. 14, each dot represents one cell being detected in thesample flow. The dots in the scatter plot fall into two distinguishedclusters. The cluster with lower scattered light intensities and higherfluorescence intensities correspond to platelets, whereas the clusterwith higher scattered light intensities and lower fluorescenceintensities correspond to red blood cells. The analysis unit enumeratesthe number of dots in each cluster to determine the total count ofplatelets and the total count of red blood cells. The analysis unit canevaluate the intensities of the scattered light of all dots in the redblood cell cluster to determine the sizes of individual red blood cellsand the size distribution of the red blood cell population in themeasurement sample. The analysis unit can also evaluate the intensitiesof the scattered light of all dots in the platelet cluster to determinethe sizes of individual platelets and the size distribution of theplatelet population in the measurement sample.

In some embodiments, a device or device system as described herein isused to analyze the red blood cells and platelets in a blood sample. Thesample supply unit prepares a measurement sample by mixing the bloodsample with a dilution reagent containing at least one compound thatadjusts the osmolality of the prepared sample. The reagent is used todilute the concentration of the red blood cells in the prepared sample,while minimizing the undesirable lysing of the red blood cells. Examplesof the osmolarity-adjusting compound include but are not limited to:salts containing cations (e.g., Na⁺, K⁺, NH₄ ⁺, Ca²⁺, and Mg²⁺containing salts); salts containing anions (e.g., Cl⁻, Br⁻, NO₃ ⁻, CO₃²⁻; HCO₃ ⁻, SO₄ ²⁻, HSO₄ ⁻, PO₄ ³⁻, HPO₄ ²⁻, H₂PO₄ ⁻, COOH⁻, andCH₃COO⁻); organic compounds such as sugars (e.g., glucose and sucrose);and alcohols (e.g., ethanol and methanol), etc. The prepared measurementsample is supplied to the flow cell of the detection unit to form asample flow. The sample flow is illuminated with an irradiation light,and the signals of the scattered light with the forward angle aremeasured in a detector. The analysis unit analyzes the detected signalsto obtain information of the measurement sample, which includes but isnot limited to one or more of the following parameters: the total countof the red blood cells, the total count of the platelets, the sizes ofindividual red blood cells, the size distribution of the red blood cellpopulation, the sizes of individual platelets, and the size distributionof the platelet population, the count of reticulocyte cells, and thecount of immature platelet cells, etc.

In a non-limiting example, the osmolarity-adjusting compound in thedilution reagent is sodium chloride. In this example, a dichroic mirrormay not be required. An obstruction bar is used as the beam stopper toblock the irradiation light. The obstruction bar has a bar width thatblocks the scattered light with a scattering angle θ₁ less than about 1degree. An aperture having a transparent opening is used in front of thedetector that receives the light comprising the scattered light, and theaperture blocks the scattered light with a scattering angle θ₂ more thanabout 7 degrees from entering the detector. The analysis unit uses thedetected signals of the scattered light to produce a histogram plot, asshown in FIG. 15. In this histogram plot, the intensities of thescattered light are plotted as the x-axis, and the numbers of thedetected cells with the corresponding intensities of the scattered lightare plotted as the y-axis. The histogram indicates two distinguishedpeaks. The peak with the lower intensities corresponds to platelets, andthe peak with higher intensities corresponds to red blood cells. Theanalysis unit enumerates the number of cells in each peak to determinethe total count of platelets and the total count of red blood cells. Theanalysis unit can evaluate the scattered light intensities from thecells in the red blood cell peak to determine the sizes of individualred blood cells and the size distribution of the red blood cellpopulation in the sample. The analysis unit can also evaluate thescattered light intensities from the cells in the platelet peak todetermine the sizes of individual platelets and the size distribution ofthe platelet population in the measurement sample.

In some embodiments, a device or device system as described herein isused to analyze the white blood cells, red blood cells and platelets ina blood sample. The sample supply unit prepares one measurement sampleby mixing one portion of the blood sample with a first staining reagentcontaining at least a first fluorescent dye. Also, the sample supplyunit prepares another measurement sample by mixing another portion ofthe blood sample with a second staining reagent containing at least asecond fluorescent dye. The first and second staining reagents can besame or different. The first and second fluorescent dyes can be same ordifferent. They can be nucleic acid dyes. Examples of fluorescent dyesinclude but are not limited to propidium iodide, ethidium bromide, DAPI,Hoechst dyes, Acridine Orange, 7-AAD, LDS 751, TOTO families of dyes,TO-PRO families of dyes, SYTO family of dyes, Thiazole Orange, BasicOrange 21, Auramine-O, and the dye compounds disclosed in U.S. Pat. No.6,004,816, etc., which is incorporated herein by reference in itsentirety as if fully set forth.

In the detection unit, one measurement sample is first supplied to theflow cell to form a first sample flow; the first sample flow isilluminated with a first irradiation light; and the signals (e.g.,fluorescence and scattered light with the forward angle) are measuredwith two detectors. After completing the measurement of the first sampleflow, another measurement sample is then supplied to the flow cell toform a second sample flow; the second sample flow is illuminated with asecond irradiation light; and the signals (e.g., fluorescence andscattered light with the forward angle) are measured with two detectors.The first and second irradiation lights can be same or different. Theanalysis unit analyzes the detected signals (e.g., fluorescence andscattered light) to obtain the information of the measurement samples,which includes but is not limited to one or more of the followingparameters: the total count of the white blood cells, the counts andpercentages of different subtypes of the white blood cells (e.g.,lymphocytes, monocytes, neutrophils, eosinophils, and basophils, etc.),the total count of the red blood cells, the total count of theplatelets, the sizes of individual red blood cells, the sizedistribution of the red blood cell population, the sizes of individualplatelets, the size distribution of the platelet population, the countof reticulocyte cells, and the count of immature platelet cells, etc.

In some embodiments, a device or device system as described herein isused to analyze white blood cells, red blood cells and platelets in ablood sample. The sample supply unit prepares a first measurement sampleby mixing a portion of the blood sample with a first reagent. The samplesupply unit further prepares a second measurement sample by mixing aportion of the first measurement sample with a second reagent. The firstreagent contains at least a fluorescent dye.

In the detection unit, the second measurement sample is first suppliedto the flow cell to form a sample flow and two light signals (e.g.,fluorescent light and scattered light with a forward angle) are measuredby two detectors. The measured signals of the fluorescent light andscattered light are used by the signal analysis unit to determine thecount of red blood cells, or the count of platelets, or both. Aftermeasuring the second measurement sample, the first measurement sample isthen supplied to the flow cell to form a sample flow and two lightsignals (e.g., fluorescent light and scattered light with a forwardangle) are measured by two detectors. The measured signals of thefluorescent light and scattered light are used by the signal analysisunit to determine the count of white blood cells, the count of differentsubtypes of white blood cells (e.g., lymphocytes, monocytes,neutrophils, eosinophils, and basophils, etc.), and the percentages ofdifferent subtypes of white blood cells (e.g., lymphocytes, monocytes,neutrophils, eosinophils, and basophils, etc.), etc. In otherembodiments, the first measurement sample can be measured in the flowcell before the second measurement sample is measured in the flow cell.

When the collecting lens is used to collect both fluorescent light andscattered light, the efficiency of collecting light signals is animportant consideration. For example, the fluorescent light usually hasa low intensity and an optimized collection efficiency is important toimprove the detection sensitivity and signal-to-noise ratio.

FIG. 16A (top view) and FIG. 16B (side view) show a non-limiting exampleof the detection unit. The measurement sample is formed into a sampleflow in the flow cell 1601. A laser diode 1602 is used as the lightsource. The focusing module comprises an aspherical lens 1603, anaperture 1604, a spherical lens 1605 and a cylindrical lens 1606. Theirradiation light emitted from the laser diode 1602 is first collimatedby the aspherical lens 1603 into a parallel light, and further focusedby the lens pair 1605 and 1606 into a light beam spot of ellipticalshape on the flow cell. The aperture 1604 is used to define the diameterof the collimated light. A collection lens 1608 is used to collect thesignal light from the flow cell into a collimated light. The detectionmodule further comprises a beam stopper 1607 between the flow cell andthe collecting lens to block the irradiation light. The receiving modulecomprises a beam splitter 1609, a focusing lens 1610, a filter 1611, asecond focusing lens 1613, and an aperture 1614. The beam splitter 1609separates the collimated signal light from the collecting lens into twooptical paths. In one path, the signal light passes through the focusinglens 1610 and the filter 1611, and then is measured by a detector 1612.In the other path, the signal light passes through the focusing lens1613 and the aperture 1614, and then is measured by a detector 1615. Bychoosing a long pass filter or band pass filter as the filter 1611, thedetector 1612 measures the intensity of the fluorescent light from thesample flow. In some embodiments, the beam splitter 1609 is a dichroicmirror. In certain embodiments, the dichroic mirror has a pass band thatmatches with the wavelength of the fluorescent light.

The selection of the collecting lens 1608 and the focusing lens 1610 isimportant to increase the collection efficiency of the fluorescent lightfrom the flow cell. First, the collecting lens 1608 limits the maximumamount of the fluorescent light that can be collected as the collimatedlight, and this maximum amount is determined by the numerical apertureof the collecting lens. Second, the selection of the collecting lens1608 and the focusing lens 1610 determines the focused spot size of thefluorescent light reaching the detector 1612. The focused spot sizedepends on the spherical aberration introduced by the lens 1608 and thelens 1610. When this focused spot size is larger than the effectivedetecting area of the detector, the portion of the fluorescent lightthat is outside the effective detecting area is not measured by thedetector. This decreases the detectable signal intensity and requires amore sensitive detector. Such an issue is particularly critical fordetectors having a small effective detecting area (e.g., photodiode,avalanche photodiode (APD), and silicon photomultipliers (SiPM)). Forexample, an aspherical lens is used as the collecting lens in order tomeasure the fluorescent light with an avalanche photodiode (APD) as thedetector (e.g., U.S. Pat. Nos. 7,894,047 and 7,580,120, which areincorporated herein by reference in their entirety as if fully setforth).

In one non-limiting example of a device or device system as describedherein, a spherical lens is used as the collecting lens 1608 and adoublet lens is used as the focusing lens 1610 to achieve an optimalcollection efficiency. A doublet lens is made of two simple lensespaired together. FIG. 17 shows a non-limiting example of a doublet lens,which comprises a first simple lens 1701 and a second simple lens 1702,which are paired together at the interface surface 1703. A doublet lensis normally used to reduce achromatic aberration, which means theaberration between different wavelengths of light. Here, a double lensis used to improve the collection efficiency of the fluorescent light ina device or device system as described herein.

FIGS. 18A and 18B show a non-limiting example demonstrating theimprovement of the collection efficiency with a doublet lens. In FIG.18A, a spherical lens 1803 is used as the collecting lens to collect thefluorescent light signal 1802 from the flow cell 1801. Another sphericallens 1804 is used as the first focusing lens to focus the collectedfluorescent light into a focused spot 1805 on the detector 1806. Incomparison, as shown in FIG. 18B, a doublet lens 1807 is used as thefirst focusing lens, and the collected fluorescent light is focused intoa focused spot 1808. The focused spot 1808 in FIG. 18B has a muchsmaller size as compared to the focused spot 1805 in FIG. 18A.Therefore, a detector with a smaller effective detection area can beused for the measurement with the doublet lens.

As shown in the non-limiting example of FIG. 16A and FIG. 16B, there areseveral surfaces in the irradiation light's path that may reflect aportion of the irradiation light back into the light source. FIG. 16C isthe zoom-in view of the light source, the focusing module and the flowcell. In this configuration, a planar surface 1619 of the spherical lens1605, a planar surface 1620 of the cylindrical lens 1606, and a planarsurface 1621 of the flow cell 1601 may each reflect a portion of theirradiation light 1617 back towards the light source 1602. If thisreflected light 1618 enters the light source, it may cause the intensityof the irradiation light to fluctuate. Some types of light source suchas a laser diode are particularly susceptible to the interference fromthe reflected light. This fluctuation issue of light source, compoundedwith the fact that a replaceable or disposable made of low-cost plasticmaterials can reflect a significant amount of the irradiation light, canmake a detection unit inaccurate for detection of particles and/orcells. Therefore, it is preferred that the reflection of the irradiationlight is eliminated or minimized. For example, the reflected light canbe directed away from the light source.

In some embodiments, the focusing module is configured to eliminate orminimize the reflection of the irradiation light from surfaces ofcomponents in the focusing module, a surface of the flow cell, or asurface in a cartridge device that hosts the flow cell. Non-limitingexamples of a cartridge device that hosts a flow cell are described inU.S. patent application Ser. No. 15/803,133 and U.S. patent applicationSer. No. 15/819,416, which are incorporated herein by reference in theirentirety as if fully set forth. The cartridge device is received into areader instrument for analysis. In some embodiments, the detectionmodule is a component of the reader instrument. As a non-limitingexample, to reduce the reflection of the irradiation light, ananti-reflection coating can be applied onto surfaces of components inthe focusing module, a surface of the flow cell, or a surface in acartridge device that hosts the flow cell.

In the configuration of FIG. 16C, the optical axis of the spherical lens1605 and the optical axis of the cylindrical lens 1606 are coaxial. Theyare further coaxial with the center axis 1616 of the irradiation lightemitted from the light source. In this configuration, the irradiationlight 1617 is reflected by the surface 1621 of the flow cell 1601, andthe reflected light 1618 is directed towards the light source 1602.

In some embodiments, the focusing module is configured to direct thereflection of the irradiation light away from the light source, or toblock the reflection of the irradiation light from entering the lightsource.

In a non-limiting example as shown FIG. 16D, the optical axis of thespherical lens 1605 and the optical axis of the cylindrical lens 1606are coaxial. However, they are not coaxial with the center axis 1616 ofthe irradiation light emitted from the light source. In this way, thereflected light 1618 is directed away from the light source and blockedby the aperture 1604 from entering the light source. FIG. 16E shows theoverview of the detection unit with the two lenses 1605 and 1606 beingcoaxial with each other, but not coaxial with center axis 1616 of theirradiation light emitted from the light source 1602.

FIG. 16F shows another non-limiting example, in which the lens 1606 iscoaxial with the center axis 1616 of the irradiation light, but the lens1605 is not coaxial with the center axis 1616 of the irradiation light.In this configuration, the reflection of the irradiation light by asurface of the flow cell or a surface in a cartridge device that hoststhe flow cell can be directed away and blocked by the aperture 1604 fromentering the light source 1602.

Other configurations can also work, if they include at least one lensnot coaxial with the irradiation light's center axis. For example, ifthe lens 1606 is not coaxial with the center axis 1616 of theirradiation light, but the lens 1605 is coaxial with the center axis1616 of the irradiation light, the reflection of the irradiation lightby a surface of the flow cell or a surface in a cartridge device thathosts the flow cell can also be directed away and blocked by theaperture 1604 from entering the light source 1602.

In various embodiments, one or more optical components in the focusingmodule are positioned not coaxial with the center axis of theirradiation light emitted from the light source. In some embodiments,such an optical component being not coaxial comprises a cylindricallens, a spherical lens, or both. In some embodiments, the optical axisof such an optical component being not coaxial is positioned away fromthe center axis of the irradiation light in the range of about 0.01 to0.1, 0.1 to 1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 mm. Invarious embodiments, an aperture is used in the focusing module to blockreflected irradiation light from entering the light source. The size oftransparent area of the aperture needs to be large enough to define thediameter of the collimated irradiation light, and small enough to blockthe reflected irradiation light. In some embodiments, the diameter ofthe transparent area of the aperture is in the range of about 0.1 to 1,1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 mm.

In some embodiments, other configurations of the focusing module canalso be used to direct the reflected irradiation light away from thelight source. A non-limiting example is shown in FIG. 16G, in which theflow cell 1601 is tilted in a way that the surface 1621 of the flow cellis not perpendicular to the irradiation light 1617. For example, theangle θ₃ between the surface 1621 and the center axis 1616 of theirradiation light 1617 is not equal to 90 degrees, and can be about45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or89-89.9 degrees. In this way, the reflected light 1618 is directed awayand blocked by the aperture 1604 from entering the light source 1602. Invarious embodiments, the flow cell or the cartridge device that hoststhe flow cell is positioned or titled in such an orientation that areflective surface of the flow cell or the cartridge device is notperpendicular to the central axis of the irradiation light and directsthe reflected light away from the light source. In some otherembodiments, a lens in the focusing module is positioned or titled insuch an orientation that the surface of the lens is not perpendicular tothe central axis of the irradiation light and directs the reflectedlight away from the light source.

Various embodiments of the present disclosure provide a device or devicesystem. The device or device system comprises: a flow cell configured toform a sample flow of a measurement sample; a light source configured toemit an irradiation light for illuminating the sample flow; a collectinglens configured to collect both a scattered light with a forward angleand a fluorescent light from the sample flow; a first light detectorconfigured to detect the collected scattered light; and a second lightdetector configured to detect the collected fluorescent light.

Various embodiments of the present disclosure provide a device or devicesystem for analyzing cells (e.g., blood cells). The device or devicesystem comprises: a flow cell configured to form a sample flow of ameasurement sample comprising cells; a light source configured to emitan irradiation light for illuminating the sample flow; a collecting lensconfigured to collect both a scattered light with a forward angle and afluorescent light from the sample flow; a first light detectorconfigured to detect the collected scattered light; and a second lightdetector configured to detect the collected fluorescent light. Invarious embodiments, the cells are blood cells. In some embodiments, thecells are white blood cells, red blood cells, or platelet cells, orcombinations thereof. In some embodiments, the cells are lymphocytes,monocytes, neutrophils, eosinophils, or basophils, or combinationsthereof. In various embodiments, the blood cells are labeled with afluorescent dye. In various embodiments, the fluorescent dye is anucleic acid dye.

In various embodiments, the irradiation light forms an elliptical beamspot on the sample flow. In various embodiments, the major axis (d₂) ofthe elliptical beam spot is perpendicular to the direction of the sampleflow and the minor axis (d₁) of the elliptical beam spot is along thedirection of the sample flow. In some embodiments, the d₂:d₁ ratio ismore than 1. In some embodiments, the d₂:d₁ ratio is about 2-5. In someembodiments, the d₂:d₁ ratio is about 5-10. In some embodiments, thed₂:d₁ ratio is about 10-15. In some embodiments, the d₂:d₁ ratio isabout 15-20. In some embodiments, the d₂:d₁ ratio is about 20-25. Insome embodiments, the d₂:d₁ ratio is about 25-40. In variousembodiments, d₂ is about 5-10, 10-15, 15-20, 20-25, 25-40, or 40-60times of the sample flow's width (d₃). In various embodiments, a deviceor device system as described herein further comprises a focusing moduleconfigured to shape the irradiation light into an elliptical beam spoton the sample flow. In some embodiments, the focusing module comprisesone cylindrical lens. In other embodiments, the focusing modulecomprises two cylindrical lenses, wherein the two cylindrical lenses areso positioned that their cylinder axes are perpendicular to each other.In still other embodiments, the focusing module comprises more than twocylindrical lenses. In various embodiments, the focusing modulecomprises a cylindrical lens, an anamorphic prism pair, or a diffractiongrating component, or combinations thereof.

In various embodiments, the collecting lens of the detection unit isused to collect both a fluorescent light and a scattered light. In someembodiments, the collecting lens of the detection unit is used tocollect both a fluorescent light and a forward scattered light (i.e., ascattered light with a forward angle (e.g., a scattering angle less thanabout 25 degrees)). In some embodiments, the collecting lens of thedetection unit is used to collect both a fluorescent light and a sidescattered light (i.e., a scattered light with a side angle (e.g., ascattering angle more than about 25 degrees)).

In various embodiments, the scattered light collected by the collectinglens comprises a scattered light with a forward angle. In variousembodiments, the scattered light collected by the collecting lenscomprises a scattered light with a scattering angle less than about 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 degrees. In various embodiments, the scattered lightdetected by the first detector comprises a scattered light with aforward angle. In various embodiments, the scattered light detected bythe first detector comprises a scattered light with a scattering angleless than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 degrees. In some embodiments, thecollected scattered light comprises light from elastic scattering. Insome embodiments, the collected scattered light comprises light fromnon-elastic scattering.

In various embodiments, the scattered light collected by the collectinglens comprises a scattered light with a side angle. In variousembodiments, the scattered light collected by the collecting lenscomprises a scattered light with a scattering angle more than about 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees. Invarious embodiments, the scattered light detected by the first detectorcomprises a scattered light with a side angle. In various embodiments,the scattered light detected by the first detector comprises a scatteredlight with a scattering angle more than about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, or 90 degrees. In some embodiments, thecollected scattered light comprises light from elastic scattering. Insome embodiments, the collected scattered light comprises light fromnon-elastic scattering.

In various embodiments, the detection unit can measure both afluorescent light and a side scattered light to analyze particles and/orcells in a sample flow. A non-limiting example of such an analysismethod is disclosed in U.S. Pat. No. 7,894,047, which is incorporatedherein by reference in its entirety as if fully set forth. In such anexample, an analyzer uses a collecting lens to collect both afluorescent light and a side scattered light, and further measures themto analyze white blood cells in a sample.

In various embodiments, the detection unit can measure both afluorescent light and a forward scattered light to analyze particlesand/or cells in a sample flow. A non-limiting example of such ananalysis method is disclosed in U.S. Pat. No. 6,004,816, which isincorporated herein by reference in its entirety as if fully set forth.

In certain embodiments, the collecting lens is one lens. In someembodiments, one collecting lens is configured to collect both ascattered light with a forward angle and a fluorescent light from thesample flow. In various embodiments, the collected scattered light andthe collected fluorescent light are detected with two separateddetectors. In various embodiments, the collected scattered light and thecollected fluorescent light are split into two separate optical paths.

In various embodiments, the second light detector comprises a bipolarphototransistor, a photosensitive field-effect transistor, aphotomultiplier tube, an avalanche photodiode, a photodiode, a CCDdevice, or a CMOS device, or combinations thereof.

In various embodiments, a device or device system as described hereinfurther comprises a receiving module configured to split the collectedscatter light and the collected fluorescent light into two separateoptical paths. In various embodiments, the receiving module comprises adichroic mirror for splitting the collected scatter light and thecollected fluorescent light into two separate optical paths. In someembodiments, the receiving module reflects the scattered light to thefirst light detector and transmits the fluorescent light to the secondlight detector. In other embodiments, the receiving module transmits thescattered light to the first light detector and reflects the fluorescentlight to the second light detector.

In some embodiments, the sample flow irradiated by the beam spot isformed in a flow cell without sheath flow (i.e., a sheathless flowcell). Examples of the sheathless flow cell include but are not limitedto those disclosed in U.S. Patent Application No. 62/497,075, U.S.patent application Ser. No. 15/803,133, and U.S. patent application Ser.No. 15/819,416, which are incorporated herein by reference in theirentirety as if fully set forth. In other embodiments, the sample flowirradiated by the beam spot is formed in a flow cell with sheath flow.

In some embodiments, the irradiation light is a Gaussian beam. Invarious embodiments, the light source comprises a laser diode, a LEDdevice, or a halogen lamp, or combinations thereof. In variousembodiments, the light source comprises: a light-emitting componentconfigured to emit a light; an optical fiber; and a condenser lensconfigured to focus the light into one end of the optical fiber, wherebythe light exits the other end of the optical fiber. In variousembodiments, the light-emitting component comprises a laser diode, a LEDdevice, or a halogen lamp, or combinations thereof. In some embodiments,the optical fiber is a single-mode optical fiber.

In various embodiments, a device or device system as described hereinfurther comprises a beam stopper between the sample flow and thecollecting lens, wherein the beam stopper is configured to block theirradiation light. In various embodiments, a device or device system asdescribed herein further comprises a beam stopper behind the collectinglens, wherein the beam stopper is configured to block the irradiationlight.

In various embodiments, a device or device system as described hereinfurther comprises an aperture on the scattered optical path from thesample flow to the first light detector, wherein the aperture isconfigured to limit the scattered light entering the first lightdetector.

Various embodiments of the present disclosure provide a method. Thismethod comprises: forming a sample flow of a measurement sample using aflow cell; illuminating the sample flow using an irradiation lightemitted from a light source; collecting both a scattered light with aforward angle and a fluorescent light from the sample flow using acollecting lens; detecting the collected scattered light using a firstlight detector; and detecting the collected fluorescent light using asecond light detector. In some embodiments, this method is performedtwice or more times using the same flow cell. In certain embodiments,the collecting lens is one lens. Various embodiments of the presentdisclosure provide a method. This method, comprises: forming a firstsample flow of a first measurement sample using a flow cell;illuminating the first sample flow using an irradiation light emittedfrom a light source; collecting both a scattered light with a forwardangle and a fluorescent light from the first sample flow using acollecting lens; detecting the collected scattered light using a firstlight detector; detecting the collected fluorescent light using a secondlight detector; forming a second sample flow of a second measurementsample using the same flow cell; illuminating the second sample flowusing the irradiation light emitted from the light source; collectingboth a scattered light with a forward angle and a fluorescent light fromthe second sample flow using the collecting lens; detecting thecollected scattered light using a first light detector; and detectingthe collected fluorescent light using a second light detector. Incertain embodiments, the collecting lens is one lens.

Various embodiments of the present disclosure provide a method foranalyzing cells (e.g., blood cells). This method comprises: forming asample flow of a measurement sample comprising cells using a flow cell;illuminating the sample flow using an irradiation light emitted from alight source; collecting both a scattered light with a forward angle anda fluorescent light from the sample flow using a collecting lens;detecting the collected scattered light using a first light detector;and detecting the collected fluorescent light using a second lightdetector. In certain embodiments, the collecting lens is one lens. Invarious embodiments, the cells are blood cells. In some embodiments, thecells are white blood cells, red blood cells, or platelet cells, orcombinations thereof. In some embodiments, the cells are lymphocytes,monocytes, neutrophils, eosinophils, or basophils, or combinationsthereof. In various embodiments, the blood cells are labeled with afluorescent dye. In certain embodiments, the fluorescent dye is anucleic acid dye. In some embodiments, this method is preformed twice ortimes using the same flow cell.

Various embodiments of the present disclosure provide a method foranalyzing cells (e.g., blood cells). This method, comprises: forming afirst sample flow of a first measurement sample comprising cells using aflow cell; illuminating the first sample flow using an irradiation lightemitted from a light source; collecting both a scattered light with aforward angle and a fluorescent light from the first sample flow using acollecting lens; detecting the collected scattered light using a firstlight detector; detecting the collected fluorescent light using a secondlight detector; forming a second sample flow of a second measurementsample comprising cells using the same flow cell; illuminating thesecond sample flow using the irradiation light emitted from the lightsource; collecting both a scattered light with a forward angle and afluorescent light from the second sample flow using the collecting lens;detecting the collected scattered light using a first light detector;and detecting the collected fluorescent light using a second lightdetector. In certain embodiments, the collecting lens is one lens.

In various embodiments, the cells in the first measurement sample arelabeled with a fluorescent dye. In certain embodiments, the fluorescentdye is a nucleic acid dye. In various embodiments, the cells in thefirst measurement sample are blood cells. In some embodiments, the cellsin the first measurement sample are white blood cells, red blood cells,or platelet cells, or combinations thereof. In some embodiments, thecells in the first measurement sample are lymphocytes, monocytes,neutrophils, eosinophils, or basophils, or combinations thereof. Invarious embodiments, the blood cells in the first measurement sample arelabeled with a fluorescent dye. In various embodiments, the cells in thesecond measurement sample are labeled with a fluorescent dye. In certainembodiments, the fluorescent dye is a nucleic acid dye. In variousembodiments, the cells in the second measurement sample are blood cells.In some embodiments, the cells in the second measurement sample arewhite blood cells, red blood cells, or platelet cells, or combinationsthereof. In some embodiments, the cells in the second measurement sampleare lymphocytes, monocytes, neutrophils, eosinophils, or basophils, orcombinations thereof.

In some embodiments, a method as described herein further comprisespreparing a measurement sample by mixing a sample with a first stainingand/or dilution reagent.

In various embodiments, a method as described herein further comprisesusing the detected signals of the scattered light and/or the fluorescentlight to analyze the cells (e.g., blood cells) in the measurementsample.

In various embodiments, a method as described herein further comprisessplitting the collected scattered light and the collected fluorescentlight into two separate optical paths.

In various embodiments, a method as described herein further comprisesblocking the irradiation light using a beam stopper between the sampleflow and the collecting lens. In various embodiments, a method asdescribed herein further comprises blocking the irradiation light usinga beam stopper behind the collecting lens.

In various embodiments, the measurement sample comprises cells, orparticles, or combinations thereof. Examples of the cells include butare not limited to blood cells. Examples of the particles include butare not limited to liquid droplets, molecules (e.g., nucleic acidmolecules, protein molecules, etc.), viruses, and beads. In someembodiments, the measurement sample comprises white blood cells, redblood cells, or platelet cells, or combinations thereof. In someembodiments, the measurement sample comprises lymphocytes, monocytes,neutrophils, eosinophils, or basophils, or combinations thereof. In someembodiments, the measurement sample comprises liquid droplets, molecules(e.g., nucleic acid molecules, protein molecules, etc.), viruses, orbeads, or combinations thereof. In various embodiments, the cells arelabeled with a fluorescent dye. In various embodiments, the particlesare either fluorescent or labeled with a fluorescent dye. In variousembodiments, a method as described herein further comprises labeling thecells with a fluorescent dye.

Various embodiments of the present disclosure provide a device or devicesystem comprising: a flow cell for forming a sample flow from ameasurement sample; a light source for emitting an irritation light toirradiate the measurement sample in the flow cell; a focusing module forfocusing the irradiation light into a beam spot of elliptical shape onthe flow cell; a collecting lens for collecting both the fluorescenceand the scattered light from the measurement sample; a receiving modulefor splitting the collected light into at least two optical paths,wherein the light in one optical path comprises the scattered light andis detected by a first detector, and the light in another optical pathcomprise the fluorescence and is detected by a second detector; and ananalysis unit that analyzes the signals from the first and/or seconddetectors to obtain information of the measurement sample. In variousembodiments, the analyzed signals include at least one of the twosignals: the signal detected by the first detector and the signaldetected by the second detector. In various embodiments, the measurementsample is a blood sample.

The range of the scattered light detected by the first detector can beselected, for example, by adjusting the configuration of the collectinglens (e.g., its size, its distance from the sample flow, and itsorientation in relation to the direction of the irradiation light). Invarious embodiments, the collecting lens is positioned substantiallyfacing the direction of the irradiation light. In various embodiments,the scattered light detected by the first detector comprises a scatteredlight with a forward angle (i.e., a forward-angle scattered light). Invarious embodiments, the scattered light detected by the first detectorcomprises a scattered light with a scattering angle less than about 3degrees. In various embodiments, the scattered light detected by thefirst detector comprises a scattered light with a scattering angle lessthan about 5 degrees. In various embodiments, the scattered lightdetected by the first detector light comprises a scattered light with ascattering angle less than about 10 degrees. In various embodiments, thescattered light detected by the first detector comprises a scatteredlight with a scattering angle less than about 15 degrees. In variousembodiments, the scattered light detected by the first detectorcomprises a scattered light with a scattering angle less than about 20degrees.

In various embodiments, the elliptical beam spot on the flow cell has adiameter of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-99, or 99-100 μm in the direction parallel to thesample flow, and a diameter of about 40-100, 100-500, 500-1000,1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000,4000-4500, or 4500-5000 μm in the direction perpendicular to the sampleflow. In various embodiments, the sample flow formed in the flow cellhas a width of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60,60-70, 70-80, 80-90, or 90-100 μm in the direction perpendicular to thesample flow.

In some embodiments, the sample flow irradiated by the beam spot isformed in a flow cell without sheath flow (i.e., a sheathless flowcell). Examples of the sheathless flow cell include but are not limitedto those disclosed in U.S. Patent Application No. 62/497,075, U.S.patent application Ser. No. 15/803,133, and U.S. patent application Ser.No. 15/819,416, which are incorporated herein by reference in theirentirety as if fully set forth. In other embodiments, the sample flowirradiated by the beam spot is formed in a flow cell with sheath flow.

In various embodiments, the focusing module comprises at least onecylindrical lens. In various embodiments, the focusing module comprisesan anamorphic prism pair.

In various embodiments, the collecting lens is an aspherical lens. Invarious embodiments, the device or device system further comprises abeam stopper for blocking the irradiation light. In various embodiments,the beam stopper is positioned in the optical path from the collectinglens to one of the two detectors. In various embodiments, the beamstopper is positioned in the optical path from the flow cell to thecollecting lens. In various embodiments, the beam stopper is anobstruction bar comprising light blocking material. In variousembodiments, the obstruction bar has a bar width that blocks theirradiation light and the scattered light with a scattering angle lessthan about 1, 3, or 5 degrees.

In various embodiments, the light source comprises a laser diode, a LEDdevice, or a halogen lamp. In various embodiments, the light sourcecomprises a light-emitting component configured to emit a light, anoptical fiber, and a condenser configured to focus the light into oneend of the optical fiber, whereby the light exiting from the other endof the optical fiber is capable of irradiating the measurement sample.In various embodiments, the light-emitting component is a laser diode, aLED device, or a halogen lamp. In various embodiments, the detector fordetecting the fluorescence comprises a photodiode. In variousembodiments, the irradiation light is a Gaussian beam.

In various embodiments, a device or device system as described hereinfurther comprises a sample supply unit configured to prepare ameasurement sample by mixing a sample with a staining and/or dilutionreagent. In various embodiments, the dilution reagent comprises at leastone osmolarity-adjusting compound. In various embodiments, the stainingreagent comprises at least one fluorescent dye. In various embodiments,the fluorescent dye is a nucleic acid dye, which selectively bind tonucleic acids. In various embodiments, the staining reagent furthercomprises a lysing compound that lyses red blood cells. In variousembodiments, the staining reagent further comprises a sphering compoundthat spherizes red blood cells. In various embodiments, the dilutionreagent further comprises a sphering compound that spherizes red bloodcells. In various embodiments, the sample is a blood sample.

In various embodiments, a device or device system as described hereincan be used to analyze blood samples and obtain information on bloodcells in the blood sample. In various embodiments, the analysis unitclassifies the white blood cells in the measurement sample into one ormore subtypes including but not limited to lymphocytes, monocytes,neutrophils, eosinophils, and basophils. In various embodiments, theanalysis unit classifies the blood cells in the measurement sample intored blood cells and platelets.

Various embodiments for the present disclosure provide a method foranalyzing a blood sample. The method comprises: preparing at least onemeasurement sample by mixing a portion or the whole of the blood samplewith a staining and/or dilution reagent; forming a sample flow of themeasurement sample in a flow cell; focusing an irradiation light from alight source into an elliptical beam spot on the sample flow in the flowcell; collecting both the fluorescence and the scattered light from themeasurement sample using one collecting lens; splitting the collectedlight from the collecting lens into at least two optical paths, whereinthe light in one optical path comprise the scattered light and isdetected by a first detector, and the light in another optical pathcomprises the fluorescence and is detected by a second detector; andanalyzing the signals from the detectors to obtain information of theblood cells in the measurement sample. In various embodiments, theanalyzed signals include at least one of the two signals: the signaldetected by the first detector and the signal detected by the seconddetector. In various embodiments, the scattered light detected by thefirst detector comprises a scattered light with a forward angle (i.e., aforward-angle scattered light). In various embodiments, the scatteredlight detected by the first detector comprises a scattered light with ascattering angle less than about 3 degrees. In various embodiments, thescattered light detected by the first detector comprises a scatteredlight with a scattering angle less than about 5 degrees. In variousembodiments, the scattered light detected by the first detector lightcomprises a scattered light with a scattering angle less than about 10degrees. In various embodiments, the scattered light detected by thefirst detector comprises a scattered light with a scattering angle lessthan about 15 degrees. In various embodiments, the scattered lightdetected by the first detector comprises a scattered light with ascattering angle less than about 20 degrees.

In various embodiments, the method further comprises classifying theblood cells into one or more cell types including but not limited towhite blood cells, red blood cells, and platelets. In some embodiments,the method further comprises classifying the blood cells into red bloodcells and platelets. In various embodiments, the method furthercomprises classifying the white blood cells in one measurement sampleinto one or more subtypes including but not limited to lymphocytes,monocyte, neutrophils, eosinophils, and basophils. In variousembodiments, the red blood cells are lysed, for example, in themeasurement sample prepared for analyzing the white blood cells. Invarious embodiments, the method further comprises preparing at least onemeasurement sample by mixing a portion or the whole of the blood samplewith a lysing compound, whereby the red blood cells are lysed in themeasurement sample. In various embodiments, the method further comprisespreparing at least one measurement sample by mixing a portion or thewhole of the blood sample with a sphering compound, whereby the redblood cells are spherized in the measurement sample.

In various embodiments, a method described herein comprises: preparing afirst measurement sample by mixing blood with a first staining and/ordilution reagent; forming a first sample flow of the first measurementsample in the flow cell; analyzing the signals from the detectors toobtain information of the while blood cells in the first measurementsample; preparing a second measurement sample by mixing blood with asecond staining and/or dilution reagent; forming a second sample flow ofthe second measurement sample in the flow cell; and analyzing thesignals from the detectors to obtain information of the red blood cellsand platelets in the first measurement sample, wherein the first andsecond samples flows are formed separately in the flow cell. In variousembodiments, the obtained information of the white blood cells includesat least one of the following parameters: the number of total whiteblood cells, the number of lymphocyte cells, the number of monocytecells, the number of neutrophil cells, the number of eosinophil cells,and the number of basophil cells. In various embodiments, the obtainedinformation of the red blood cells and platelets includes at least oneof the following parameters: the number of red blood cells, the numberof platelets, the sizes of individual red blood cells, the sizedistribution of the red blood cell population, the sizes of individualplatelets, the size distribution of the platelet population, the numberof reticulocyte cells, and the number of immature platelet cells, etc.

In accordance with the present disclosure, the terms “first” and“second” are used to designate identities but not to indicate anychronological sequence.

Many variations and alternative elements have been disclosed inembodiments of the present disclosure. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of fluidicunits, components and structures for the inventive devices and methods,and the samples that may be analyzed therewith. Various embodiments ofthe disclosure can specifically include or exclude any of thesevariations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the disclosure are tobe understood as being modified in some instances by the term “about.”As one non-limiting example, one of ordinary skill in the art wouldgenerally consider a value difference (increase or decrease) no morethan 10% to be in the meaning of the term “about.” Accordingly, in someembodiments, the numerical parameters set forth in the writtendescription and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of thedisclosure may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The disclosure is explained by various examples, which are intended tobe purely exemplary of the disclosure, and should not be considered aslimiting the disclosure in any way. Various examples are provided tobetter illustrate the claimed disclosure and are not to be interpretedas limiting the scope of the disclosure. To the extent that specificmaterials are mentioned, it is merely for purposes of illustration andis not intended to limit the disclosure. One skilled in the art maydevelop equivalent means or reactants without the exercise of inventivecapacity and without departing from the scope of the disclosure.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the disclosure are described above in theDetailed Description. While these descriptions directly describe theabove embodiments, it is understood that those skilled in the art mayconceive modifications and/or variations to the specific embodimentsshown and described herein. Any such modifications or variations thatfall within the purview of this description are intended to be includedtherein as well. Unless specifically noted, it is the intention of theinventors that the words and phrases in the specification and claims begiven the ordinary and accustomed meanings to those of ordinary skill inthe applicable art(s).

The foregoing description of various embodiments of the disclosure knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the disclosure to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe disclosure and its practical application and to enable othersskilled in the art to utilize the disclosure in various embodiments andwith various modifications as are suited to the particular usecontemplated. Therefore, it is intended that the disclosure not belimited to the particular embodiments disclosed for carrying out thedisclosure.

While particular embodiments of the present disclosure have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this disclosure and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this disclosure.

Additional Aspects of the Present Disclosure

Aspects of the subject matter described herein may be useful alone or incombination with any one or more of the other aspect described herein.Without limiting the foregoing description, in a first aspect of thepresent disclosure, a device or device system comprises: a flow cellconfigured to form a sample flow of a measurement sample, wherein themeasurement sample comprises particles and/or cells; a light sourceconfigured to emit an irradiation light for illuminating the sampleflow; a collecting lens configured to collect both a scattered lightwith a forward angle and a fluorescent light from the particles and/orcells in the sample flow; and one, two, or more detectors configured todetect a signal of the scattered light with a forward angle and a signalof the fluorescent light.

In accordance with a second aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a device or device system as described herein furthercomprises a focusing module configured to focus the irradiation light toform an elliptical beam spot on the sample flow.

In accordance with a third aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a device or device system as described herein furthercomprises a focusing module that comprises a lens that is either notcoaxial or not perpendicular with the central axis of irradiation light.

In accordance with a fourth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the collected and/or detected scattered light includes ascattered light with a scattering angle less than about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25degrees.

In accordance with a fifth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the flow cell is part of a cartridge device configured tobe placed into a reader instrument for analysis, and wherein the readerinstrument comprises a light source, a collecting lens, detectors, and asignal analysis unit.

In accordance with a sixth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the cartridge device is configured to mix a sample with areagent to form the measurement sample and to form a sample flow of themeasurement sample in the flow cell.

In accordance with a seventh aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a device or device system as described herein furthercomprises a receiving module configured to split the scattered lightwith a forward angle and the fluorescent light collected by thecollecting lens into two separate optical paths toward two separatedetectors, respectively.

In accordance with an eighth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a device or device system as described herein furthercomprises a doublet lens configured to focus the collected fluorescentlight.

In accordance with a ninth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a device or device system as described herein furthercomprise a signal analysis unit configured to analyze the signal of thescattered light with a forward angle and the signal of the fluorescentlight for analyzing the particles and/or cells.

In accordance with a tenth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a method of analyzing particles and/or cells in ameasurement sample comprises: using a flow cell to form a sample flow ofthe measurement sample; using a light source to emit an irradiationlight; using the irradiation light to illuminate the sample flow; usinga collecting lens to collect both a scattered light with a forward angleand a fluorescent light from the particles and/or cells in the sampleflow; and using one, two, or more detectors to detect a signal of thescattered light with a forward angle and a signal of the fluorescentlight.

In accordance with an eleventh aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, two separate detectors are used to detect thesignal of the scattered light with a forward angle and the signal of thefluorescent light.

In accordance with a twelfth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, a method as described herein further comprises using afocusing module to focus the irradiation light to form an ellipticalbeam spot on the sample flow.

In accordance with a thirteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the collected and/or detected scattered lightincludes a scattered light with a scattering angle less than about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 degrees

In accordance with a fourteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing a doublet lens to focus the collected fluorescent light.

In accordance with a fifteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the flow cell is part of a cartridge deviceconfigured to be placed into a reader instrument for analysis, andwherein the reader instrument comprises a light source, a collectinglens, detectors, and a signal analysis unit.

In accordance with a sixteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing the cartridge device to mix a sample with a reagent to form themeasurement sample and to form a sample flow of the measurement samplein the flow cell.

In accordance with a seventeenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing a signal analysis unit to analyze the signal of the scatteredlight with a forward angle and the signal of the fluorescent light foranalyzing the particles and/or cells.

In accordance with an eighteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, a method for analyzing particles and/cells in asample, comprising: receiving the sample into a cartridge devicecomprising a flow cell; using the cartridge device to mix the sample thesample with a reagent to form a measurement sample; using the flow cellto form a sample flow of the measurement sample; using a light source toemit an irradiation light; using the irradiation light to illuminate thesample flow; using a collecting lens to collect both a scattered lightand a fluorescent light from the particles and/or cells in the sampleflow; and using one, two, or more detectors to detect a signal of thescattered light and a signal of the fluorescent light.

In accordance with a nineteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the reagent comprises a fluorescence labelingcompound.

In accordance with a twentieth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, two separate detectors are used to detect thesignal of the scattered light and the signal of the fluorescent light.

In accordance with a twenty-first aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing a focusing module to focus the irradiation light to form anelliptical beam spot on the sample flow.

In accordance with a twenty-second aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the collected and/or detected scattered lightincludes a scattered light with a scattering angle less than about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 degrees.

In accordance with a twenty-third aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the collected and/or detected scattered lightincludes a scattered light with a scattering angle more than about 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees.

In accordance with a twenty-fourth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing a doublet lens to focus the collected fluorescent light.

In accordance with a twenty-fifth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, a method as described herein further comprisesusing a signal analysis unit to analyze the signal of the scatteredlight and the signal of the fluorescent light for analyzing theparticles and/or cells.

Many different arrangements of the various components depicted, as wellas components not shown, are possible without departing from the spiritand scope of the present disclosure. Embodiments of the presentdisclosure have been described with the intent to be illustrative ratherthan restrictive. Alternative embodiments will become apparent to thoseskilled in the art that do not depart from its scope. A skilled artisanmay develop alternative means of implementing the aforementionedimprovements without departing from the scope of the present disclosure.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations and are contemplated within the scope of the claims.Unless indicated otherwise, not all steps listed in the various figuresneed be carried out in the specific order described.

The disclosure claimed is:
 1. A device system, comprising: a flow cellconfigured to form a sample flow of a measurement sample, wherein themeasurement sample comprises particles or cells; a light sourceconfigured to emit an irradiation light for illuminating the sampleflow; a collecting lens configured to collect both a scattered lightwith a forward angle and a fluorescent light from the particles or cellsin the sample flow; and one, two, or more detectors configured to detecta signal of the scattered light with the forward angle and a signal ofthe fluorescent light; a receiving module configured to split thescattered light with a forward angle and the fluorescent light collectedby the collecting lens into two separate optical paths toward twoseparate detectors, respectively.
 2. The device system of claim 1,further comprising a focusing module configured to focus the irradiationlight to form an elliptical beam spot on the sample flow.
 3. The devicesystem of claim 1, further comprising a focusing module that comprises alens that is either not coaxial or not perpendicular with a central axisof the irradiation light.
 4. The device system of claim 1, wherein acollected or detected scattered light comprises a scattered light with ascattering angle less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees.
 5. The devicesystem of claim 1, wherein: the flow cell is part of a cartridge deviceconfigured to be placed into a reader instrument for analysis; and thereader instrument comprises the light source, the collecting lens, thedetectors, and a signal analysis unit.
 6. The device system of claim 5,wherein the cartridge device is configured to mix a sample with areagent to form the measurement sample and to form a sample flow of themeasurement sample in the flow cell.
 7. The device of claim 6, whereinthe reagent is a fluorescent labeling compound.
 8. The device system ofclaim 1, further comprising a doublet lens configured to focus thecollected fluorescent light.
 9. The device system of claim 1, furthercomprising a signal analysis unit configured to analyze the signal ofthe scattered light with a forward angle and the signal of thefluorescent light for analyzing the particles or cells.
 10. A method ofanalyzing particles or cells in a measurement sample, comprising: usinga flow cell to form a sample flow of the measurement sample; using alight source to emit an irradiation light; using the irradiation lightto illuminate the sample flow; using a collecting lens to collect both ascattered light with a forward angle and a fluorescent light from theparticles or cells in the sample flow; using one, two, or more detectorsto detect a signal of the scattered light with a forward angle and asignal of the fluorescent light; and using a receiving module configuredto split the scattered light with a forward angle and the fluorescentlight collected by the collecting lens into two separate optical pathstoward two separate detectors, respectively.
 11. The method of claim 10,wherein two separate detectors are used to detect the signal of thescattered light with a forward angle and the signal of the fluorescentlight.
 12. The method of claim 10, further comprising using a focusingmodule to focus the irradiation light to form an elliptical beam spot onthe sample flow.
 13. The method of claim 10, wherein the collected ordetected scattered light comprises a scattered light with a scatteringangle less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees.
 14. The method of claim10, wherein the collected or detected scattered light comprises ascattered light with a scattering angle more than about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees.
 15. The method ofclaim 10, further comprising using a doublet lens to focus the collectedfluorescent light.
 16. The method of claim 1, wherein: the flow cell ispart of a cartridge device configured to be placed into a readerinstrument for analysis; and the reader instrument comprises the lightsource, the collecting lens, the detectors, and a signal analysis unit.17. The method of claim 16, further comprising using the cartridgedevice to mix a sample with a reagent to form the measurement sample andto form a sample flow of the measurement sample in the flow cell. 18.The method of claim 17, wherein the reagent is a fluorescence labelingcompound.
 19. The method of claim 10, further comprising using a signalanalysis unit to analyze the signal of the scattered light with aforward angle and the signal of the fluorescent light for analyzing theparticles or cells.